Microwell array articles and methods of use

ABSTRACT

The disclosure provides microstructured articles and methods useful for detecting an analyte in a sample. The articles include microwell arrays. The articles can be used with an optical system component in methods to detect or characterize an analyte.

BACKGROUND

The ability to perform parallel microanalysis on minute quantities ofsample is important to the advancement of chemistry, biology, drugdiscovery and medicine. Today, the traditional 1536-well microtiterplate has been surpassed by microwell arrays which have an even greaternumber of reaction chambers and use lesser amounts of reagents due toefforts focused on maximizing time, throughput, and cost efficiencies.Although there are several types of microwell arrays available,fabrication techniques used to generate high fidelity microfeatureshaving dimensions in tens of microns such as wells are frequently slowand expensive. Examples of common fabrication methods to produce thesemicrofeatures include soft lithography, lithography, preferentialetching of pre-existing arrays, milling, diamond machining, laserablation, chaotropic etching and the like. However, all of these methodssuffer from cost and capability limitations to varying degrees. Further,it is a special challenge to make articles with high densitymicrofeatures that exhibit the desired optical features that are foundin low-density microwell articles (e.g. 96-well plates). In addition,many microwell materials prove to be incompatible with the components ofbioassays and chemical reactions and result in problems such as lowsensitivity, high background signal, and lack of reproducibility. Thus,there continues to be a need for the development of improved microwellarrays.

Certain fiber optic bundles have been used to create microwell arrays.To act as an efficient waveguide, each fiber element must consist of ahigh refractive index core surrounded by a low refractive indexcladding. Selective removal of the core glass by chemical etching tocreate a microwell lowers the refractive index mismatch when theoriginal glass is replaced with a lower refractive index aqueoussolution typically used in biological assays. This diminishes thewaveguide characteristics of the fiber leading to increased lightpenetration through the cladding material. To overcome this problemlight absorbing materials, for example certain metals, have beendeposited as a thin layer on the interior sidewalls of the etchedmicrowells. In addition to optical limitations, the fiber opticmaterials are often incompatible with many reaction conditions,particularly assays which are conducted in aqueous solutions and containsensitive enzymatic reagents. Two major sources of incompatibility arethe dissolution of the fiber optic substrate into the solution containedin the reaction chamber and the chemical reaction of the fiber opticsubstrate with assay components (e.g., proteins, nucleic acids)contained in the chamber. These chemical effects are exacerbated by thehigh surface to volume ratio in each microwell. These effects tend todegrade the performance of assays and reactions conducted in the fiberoptic reaction chambers and frequently require additional processing torender the devices compatible with biological assays.

Due to technical difficulties in currently used processes forfabricating and/or coating arrays meeting these optical and chemicalrequirements, the range of assays that can be conducted in microwellarrays remains limited. Accordingly, there is a need for cost effective,high density microwell arrays that are compatible with a variety ofassay and/or reaction conditions.

SUMMARY

This disclosure relates to flexible microwell arrays comprising aflexible microstructured layer with an optically transmissive flexiblelayer coupled thereto.

In one aspect, the present disclosure provides an article. The articlecan comprise a microstructured layer with upper and lower majorsurfaces. The microstructured layer can comprise a plurality ofoptically-isolated microwells extending between the upper and lowermajor surfaces. The article further can comprise anoptically-transmissive flexible layer coupled to the lower major surfaceof the microstructured layer. Each microwell in the microstructuredlayer can comprise a top opening, a bottom opening, and at least oneside wall extending between the top opening and bottom opening. Theoptically-transmissive flexible layer can have a thickness of about 2 μmto about 50 μm.

In another aspect, the present disclosure provides an article. Thearticle can comprise a microstructured layer with upper and lower majorsurfaces. The microstructured layer can comprise a plurality ofoptically-isolated microwells extending below the upper major surface.The article further can comprise an optically-transmissive flexiblelayer coupled to the lower major surface of the microstructured layer.Each microwell in the microstructured layer can comprise an opening, anoptically-transmissive bottom wall, and at least one side wall extendingbetween the opening and the bottom wall. The bottom wall can have athickness of about 0.1 μm to about 5 μm.

In another aspect, the present disclosure provides an article. Thearticle can comprise a microstructured layer with upper and lower majorsurfaces. The microstructured layer can comprise a plurality ofoptically-isolated microwells extending below the upper major surface.The article further can comprise an optically-transmissive flexiblelayer coupled to the lower major surface of the microstructured layer.Each microwell in the microstructured layer can comprise an opening, anoptically-transmissive bottom wall, and at least one side wall extendingbetween the opening and the bottom wall. A thickness (t) can be definedby a thickness of the bottom wall plus a thickness of theoptically-transmissive flexible layer. Thickness (t) can be about 2 μmto about 55 μm.

In any of the above embodiments, the microstructured layer can comprisea colorant. In any of the above embodiments, the colorant can beselected from the group consisting of carbon black, fuchsin, carbazoleviolet, and Foron Brilliant Blue.

In any of the above embodiments, the optically-transmissive flexiblelayer can be transmissive to a selected wavelength of light. In any ofthe above embodiments, the bottom wall can be substantially transmissiveto the selected wavelength of light. In some embodiments, the at leastone sidewall can be substantially nontransmissive to the selectedwavelength of light. In some embodiments, the at least one sidewall canbe at least 50% less transmissive of a selected wavelength of light thanthe bottom wall. In some embodiments, the at least one sidewall can beat least 90% less transmissive of a selected wavelength of light thanthe bottom wall.

In any of the above embodiments, the bottom wall and/or the at least oneside wall of a microwell further can comprise a coating. In someembodiments, the bottom wall and/or the at least one side wall of amicrowell further comprise a plurality of coatings. In any of the aboveembodiments, the coating can comprise SiO₂. In any of the aboveembodiments, the coating can comprise a reflective coating.

In any of the above embodiments, microstructured layer can comprise acured polymer derived from a resin. In some embodiments, the resin canbe selected from the group consisting of acrylic-based resins derivedfrom epoxies, polyesters, polyethers, and urethanes; ethylenicallyunsaturated compounds; aminoplast derivatives having at least onependant acrylate group; polyurethanes (polyureas) derived from anisocyanate and a polyol (or polyamine); isocyanate derivatives having atleast one pendant acrylate group; epoxy resins other than acrylatedepoxies; and mixtures and combinations thereof.

In any of the above embodiments, the optically-transmissive layer cancomprise a film. In some embodiments, the film can comprise polyethyleneterephthalate, polyethylene naphthalate, high density polyethylene, lowdensity polyethylene, or linear low density polyethylene. In any of theabove embodiments, the film can comprise a multilayer film. In any oneof the above embodiments, the optically-transmissive layer further cancomprise an adhesive.

In any of the above embodiments, the microstructured layer further cancomprise a region that is substantially free of microwells. In someembodiments, the region can comprise a detachable portion.

In any of the above embodiments, the article further can comprise acover layer coupled to the upper major surface of the microstructuredlayer. In any one of the above embodiments, the article further cancomprise a cover layer coupled to the optically-transmissive flexiblelayer on a surface opposite the microstructured layer. In any of theabove embodiments, the cover layer may be removably coupled.

In any of the above embodiments, the article further can comprise anoptical detection system comprising an optical device optically coupledto the article. In some embodiments, the optical device can be opticallycoupled to the microstructured layer. In some embodiments, the opticaldevice can be optically coupled to the optically-transmissive flexiblelayer. In some embodiments, the coupling can comprise a fiber optic faceplate. In some embodiments, the optical device can comprise a CCD imagesensor, a CMOS image sensor, or a photomultiplier tube. In someembodiments, the optical system further can comprise a processor.

In any of the above embodiments, at least one microwell further cancomprise a polynucleotide. In any of the above embodiments, at least onemicrowell can comprise a polypeptide. In any of the above embodiments,at least one microwell can comprise a particle. In some embodiments, apolynucleotide or a polypeptide can be coupled to the particle.

In another aspect, the present disclosure provides a process formanufacturing a microwell array article. The process can compriseproviding a tool having a molding surface with a plurality ofprojections extending therefrom suitable for forming the microstructureelements; a flowable resin composition; and an optically-transmissiveflexible layer having first and second major surfaces. The first majorsurface of the optically-transmissive flexible layer can besurface-treated to promote adhesion to a cured resin composition. Thethickness of the optically-transmissive flexible layer can be about 50μm or less. The process further can comprise applying to the moldingsurface a volume of the resin composition suitable for formingmicrostructure elements. The process further can comprise contacting theresin composition with a first major surface of theoptically-transmissive flexible layer. The process further can comprisecuring the resin while it is in contact with the flexible layer to forma microwell array article comprising a microstructured layer including amicrowell array bonded to the flexible layer. The process further cancomprise removing the microwell array article from the tool.

In any of the above embodiments of the process, contacting the resincomposition with the first major surface of the optically-transmissiveflexible layer can comprise applying pressure to the resin compositionto substantially displace the resin between the tops of the projectionsin the tool and the surface of the optically-transmissive layer. In anyof the above embodiments of the process, the flexible layer can besurface-treated to promote adhesion to the cured resin composition.Surface treatments can be selected from the group consisting ofradiation treatments, corona discharge treatment, flame treatment,plasma treatment, high energy UV treatment, and chemical primingtreatment. In any of the above embodiments of the process, the flexiblelayer can be coupled to a carrier. In any of the above embodiments ofthe process, the resin composition can comprise a colorant. In any ofthe above process embodiments, curing the resin composition can compriseexposing the resin composition to at least one curing treatment selectedfrom the group consisting of actinic radiation from a radiation source,an electron beam, and a chemical curing agent. In any of the aboveembodiments of the process, the flexible layer can have a thickness ofabout 2 μm to about 48 μm. In any of the above embodiments, the processcan further comprise the step of removing a portion of themicrostructured layer. In any of the above embodiments, the process canfurther comprise the step of removing a portion of theoptically-transmissive flexible layer. In any of the above embodiments,the process further can comprise disposing a reagent in a microwell. Inany of the above embodiments of the process, the process further cancomprise the step of coupling the microwell array article to a fiberoptic device.

In another aspect, the present disclosure provides a method of detectingan analyte in a microarray. The method can comprise providing a samplesuspected of containing an analyte, a reagent for the optical detectionof the analyte, an optical detection system, and an article according toany of the above embodiments. The method further can comprise contactingthe sample and the reagent in a plurality of microwells under conditionssuitable to detect the analyte, if present, in a microwell. The methodfurther can comprise using the optical system to detect the presence orabsence of the analyte in a microwell.

In some embodiments of the method, the optical system can be opticallycoupled to the substrate. In some embodiments, the optical coupling cancomprise a fiber optic face plate, wherein using the optical detectionsystem can comprise passing a signal through the fiber optic face plate.In any of the above embodiments of the method, the optical system cancomprise an optical device. In some embodiments, the optical device cancomprise a CCD image sensor, a CMOS image sensor, or a photomultipliertube. In any of the above embodiments of the method, the optical systemfurther can comprise a processor.

In any of the above method embodiments, detecting the presence orabsence of an analyte can comprise detecting light that is indicative ofthe presence of the analyte. In any of the above method embodiments,detecting light can comprise detecting light by absorbance, reflectance,or fluorescence. In any of the above method embodiments, detecting lightcan comprise detecting light from a lumigenic reaction.

In any of the above method embodiments, detecting the presence orabsence of the analyte can comprise obtaining an image of a microwell.In some embodiments, detecting the presence or absence of the analytecan comprise displaying, analyzing, or printing the image of amicrowell.

In any of the above method embodiments, contacting the sample and thereagent in a plurality of microwells under conditions suitable to detectthe analyte can comprise contacting an enzyme and an enzyme substrate.In any of the above method embodiments, contacting the sample and thereagent in a plurality of microwells under conditions suitable to detectthe analyte can comprise forming a hybrid between two polynucleotides.

In another aspect, the present disclosure provides an assay system. Thesystem can comprise a microwell array article comprising an array ofoptically-isolated microwells and an imaging device coupled thereto. Thearray density can be ten or more microwells per square millimeter. Insome embodiments, optically coupled can comprise adhesively coupling themicrowell array article to a solid interface.

In another aspect, the present disclosure provides a composition. Thecomposition can comprise a compound selected from the group consistingof 1-(3-methyl-n-butylamino)-9,10-anthracenedione;1-(3-methyl-2-butylamino)-9,10-anthracenedione;1-(2-heptylamino)-9,10-anthracenedione;1,1,3,3-tetramethylbutyl-9,10-anthracenedione;1,10-decamethylene-bis-(-1-amino-9,10-anthracenedione);1,1-dimethylethylamino-9,10-anthracenedione; and1-(n-butoxypropylamino)-9,10-anthracenedione. In some embodiments, thecomposition further can comprise a cured polymer. In any of the aboveembodiments, the cured polymer can be is derived from a resin selectedfrom the group consisting of acrylate resins, acrylic resins,acrylic-based resins derived from epoxies, polyesters, polyethers, andurethanes; ethylenically unsaturated compounds; aminoplast derivativeshaving at least one pendant acrylate group; polyurethanes (polyureas)derived from an isocyanate and a polyol (or polyamine); isocyanatederivatives having at least one pendant acrylate group; epoxy resinsother than acrylated epoxies; and mixtures and combinations thereof.

The words “preferred” and “preferably” refer to embodiments of thedisclosure that may afford certain benefits, under certaincircumstances. However, other embodiments may also be preferred, underthe same or other circumstances. Furthermore, the recitation of one ormore preferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the disclosure.

As used herein, “a,” “an,” “the,” “at least one,” and “one or more” areused interchangeably. Thus, for example, a substrate comprising “an”array can be interpreted to mean that the substrate can include “one ormore” arrays.

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

The above summary of the disclosure is not intended to describe eachdisclosed embodiment or every implementation of the disclosed articles,processes, and methods. The description that follows more particularlyexemplifies illustrative embodiments. In several places throughout theapplication, guidance is provided through lists of examples, whichexamples can be used in various combinations. In each instance, therecited list serves only as a representative group and should not beinterpreted as an exclusive list.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be further explained with reference to the drawingfigures listed below, where like structure is referenced by likenumerals throughout the several views.

FIG. 1 is a top perspective exploded view, of one embodiment of amicrowell array article according to the present disclosure.

FIG. 2A is a cross-sectional view of one embodiment of a microwell arrayarticle.

FIG. 2B is a cross-sectional view showing detail of a portion of themicrowell array article of FIG. 2A.

FIG. 3A is a cross-sectional schematic view of one embodiment of amicrowell array article, with a relatively thin optical path extendingfrom the microwells to an optical device.

FIG. 3B is a cross-sectional schematic view of one embodiment of amicrowell array article, with a relatively thick optical path extendingfrom the microwells to an optical device.

FIG. 4 is a top perspective view of an embodiment of a microwell arrayarticle comprising a tab portion according to the present disclosure.

FIG. 5 is a schematic side view of one embodiment of a process formaking a microwell array article according to the present disclosure.

FIG. 6 a is a schematic top view of a microwell array article, showingthe line along which an image of the microwell array article wasanalyzed.

FIG. 6 b is a graph of the pixel intensities from the image of themicrowell array article of FIG. 6 a.

FIG. 7 is a graph of the pixel intensities from the image of anothermicrowell array.

FIG. 8A is a side view of one embodiment of a carrier article with aflexible microwell array according to the present disclosure.

FIG. 8B is a top view of the carrier article of FIG. 8A.

FIGS. 9A-9F are side views of the steps of a process to prepare amicrowell array for microanalyses according to the present disclosure.

FIG. 10 is a u.v.-visible spectrogram of a mixture of six colorants.

DETAILED DESCRIPTION

The present disclosure provides array articles comprising a substratecontaining individual reaction chambers (microwells). The disclosureincludes the process of fabricating the array including methods offorming multilayered structures (e.g., laminates) comprising thesubstrate in which the array is formed, a flexible,optically-transmissive layer coupled thereto and, optionally, one ormore removable protective layers. The inventors have discovered that itis particularly difficult to make flexible substrates with high-densitymicrofeatures (e.g., reaction chambers) that are optically isolated inboth axes of an X-Y plane, but remain optically transmissive in thecorresponding Z axis. The inventors further have discovered that it isdifficult to fabricate an array of optically-isolated microwells with athin, flexible, highly-transmissive base. The present disclosureprovides microwell arrays, and a process for making the microwellarrays, that overcome these difficulties. The inventive processes resultin significant optical isolation of the individual reaction wellswithout the need for difficult, costly coating procedures to obtain therequisite optical isolation for extremely high-density microwell arrays.

The present disclosure further provides a system to detect an analyte.The detection system can comprise any of the array articles disclosedherein, wherein the array article is optically coupled to an opticaldevice. The optical device may be an imaging device.

The disclosed articles can be used in an assay to detect an analyte. Theassay can include the detection of a fluorescent or a luminescent signalemanating from a particular microwell in the array. The disclosedarticles substantially prevent the transmission of selected wavelengthsof light from one microwell to another through the sidewalls of themicrowells. Simultaneously, the articles permit the transmission ofsubstantial amounts of the selected wavelengths of light through thebottom wall of each microwell. Even further, the disclosed articles areconfigured to minimize the scatter of light emanating from the bottom ofeach microwell such that the light emanating from individual adjacentmicrowells in an array; in particular, a high-density array; can beresolved by a simple optical device.

Before any embodiments of the disclosure are explained in detail, it isto be understood that the disclosure is not limited in its applicationto the details of construction and the arrangement of components setforth in the following description or illustrated in the accompanyingdrawings. The invention is capable of other embodiments and of beingpracticed or of being carried out in various ways. Also, it is to beunderstood that the phraseology and terminology used herein is for thepurpose of description and should not be regarded as limiting. The useof “including,” “comprising,” “containing,” or “having” and variationsthereof herein is meant to encompass the items listed thereafter andequivalents thereof as well as additional items. Unless specified orlimited otherwise, the terms “supported,” and “coupled” and variationsthereof are used broadly and encompass both direct and indirect supportsand couplings. It is to be understood that other embodiments may beutilized and structural or logical changes may be made without departingfrom the scope of the present disclosure. Furthermore, terms such as“front,” “rear,” “top,” “bottom,” and the like are only used to describeelements as they relate to one another, but are in no way meant torecite specific orientations of the apparatus, to indicate or implynecessary or required orientations of the apparatus, or to specify howthe articles described herein will be used, mounted, displayed, orpositioned in use.

The present disclosure is generally directed to methods and articles fordetecting analytes in a sample. More particularly, the disclosurerelates to microwell arrays that can be used to conduct multiple,independent assays simultaneously. The disclosed articles are adaptedfor use with a variety of optical systems to detect the presence orabsence of an analyte in a sample.

DEFINITIONS

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe relevant art to. Methods and materials similar or equivalent tothose described herein can be used in the practice of the presentdisclosure, and exemplified suitable methods and materials are describedbelow. For example, methods may be described which comprise more thantwo steps. In such methods, not all steps may be required to achieve adefined goal and the invention envisions the use of isolated steps toachieve these discrete goals. The disclosures of all publications,patent applications, patents and other references are incorporatedherein by reference in their entirety. In addition, the materials,methods, and examples are illustrative only and not intended to belimiting.

“Analyte” means a molecule, compound, composition or complex, eithernaturally occurring or synthesized, to be detected or measured in orseparated from a sample of interest. Analytes include, withoutlimitation, polypeptides (e.g., proteins), peptides, amino acids, fattyacids, nucleotides (e.g., ATP), polynucleotides (including, but notlimited to DNA, RNA, cDNA, mRNA, PNA, LNA), carbohydrates, hormones,steroids, compounds, lipids, vitamins, bacteria, viruses,pharmaceuticals, and metabolites. An analyte may be one member of aligand/anti-ligand pair or one member of a pair of polynucleotideshaving sufficient complementarity to participate in a hybridizationevent.

“Fiber optic faceplate” refers to a bundle of optical quality glassfibers which are fused together to form a monolithic structure which isthen “sliced” and polished to form a “wafer” of required thickness.

“Functional groups” means any chemical or biological species capable ofaffixing a reactant or analyte to the inside surface of the reactionchamber.

“Impermeable to water” refers to the ability of a thin film to provide abarrier to an aqueous solution contained in the reaction chamber and toprevent leaching of the chamber solution into the wall components of thereaction chamber.

“Optically transparent” refers to the ability of light to transmitthrough a material. “Optically isolated”, as used herein, refers to acondition whether by light that is directed into a microwell in anarticle or that is emitted by a component or a reaction contained in amicrowell, is not substantially transmitted laterally through thearticle and detectably associated with a proximate microwell (i.e., lessthan 20% of the light; preferably, less than 10% of the light; morepreferably, less than 5% of the light; even more preferably, less than1% of the light is transmitted and detectably associated with aproximate microwell).

“Reactant” means any chemical or biological molecule, compound,composition or complex, either naturally occurring or synthesized, thatis capable of binding, forming, or reacting with an analyte in a sampleof interest either alone or in conjunction with another reactant. Thereactants of the present disclosure are useful for chemical reaction orbiochemical measurement, detection or separation. Examples of reactantsinclude, without limitation, amino acids, nucleic acids, includingoligonucleotides and cDNA, carbohydrates, and proteins such as enzymesand antibodies.

“Reaction Chamber” means a localized well or chamber (i.e. ahollowed-out space, having width and depth) on a substrate, comprisingside walls and a bottom that is used to facilitate the interaction ofreactants.

“Scanning Electron Microscopy” or “SEM” refers to a method for highresolution imaging.

“Substrate” refers to a solid support or any material that can bemodified to contain discrete individual reaction chambers and isamenable to at least one detection method.

“Enzyme substrate” refers to a molecule that participates in anenzyme-catalyzed reaction.

“Thin film” refers to the coating of material deposited on the surfaceof the substrate less than 1 micron thick.

An “array of regions on a solid support” is a linear or two-dimensionalarray of preferably discrete regions, each having a finite area, formedon the surface of a solid support.

A “microwell array” is an array of reaction chambers having a density ofdiscrete reaction chambers of at least about 100/cm², and preferably atleast about 10/mm². The reaction chambers have a three-dimensionalstructure with dimensions, e.g., openings with, for example, diametersin the range of between about 5-250 μm, depths in the range betweenabout 2 to 100 microns. By “array” herein is meant a plurality ofreaction chambers, which are localized wells or chambers in an arrayformat on the substrate material; the size of the array and its reactionchambers will depend on the composition and end use of the array.

The term “hybridization” refers to a process in which a single-strandedregion of a nucleic acid molecule (DNA or RNA) is joined with acomplementary single-stranded region of nucleic acid, again DNA or RNA,to form a double-stranded region. Hybridization includes intermolecular(between two distinct molecules) and intramolecular (between two regionsof a single molecule) processes

“Microspheres” or “beads” or “particles” or grammatical equivalentsherein refer to small, discrete particles.

The term “probe” refers to poly peptide or a single-stranded nucleicacid molecule, with a known nucleotide sequence, which is labeled insome way (for example, radioactively, fluorescently, or immunologically)and used to selectively find and mark certain polypeptide, DNA, or RNAsequences of interest to a researcher by binding or hybridizing to it.

The term “cDNA” refers to DNA synthesized from an RNA template usingreverse transcriptase.

The term “nucleotide” refers to a subunit of DNA or RNA consisting of anitrogenous base (adenine, guanine, thymine, or cytosine in DNA;adenine, guanine, uracil, or cytosine in RNA), a phosphate molecule, anda sugar molecule (deoxyribose in DNA and ribose in RNA).

The term “oligonucleotide” refers to a compound comprising a nucleotidelinked to phosphoric acid. When polymerized, it gives rise to a nucleicacid.

The term “biomolecule” refers to an organic molecule and especially amacromolecule (as a protein or nucleic acid) that may be found in and/orsynthesized by a living organisms.

The term “labeling” refers to attachment of a moiety to a macromoleculethat enables it to be visualized or its presence detected using specificinstrumentation.

The term “nucleic acid” refers to any of various acids (as an RNA or aDNA) composed of nucleotide chains.

The term “PNA (peptide nucleic acid)” refers to Peptide nucleic acid(PNA) monomers have a N-(2-aminoethyl)glycine backbone to which adenine,cytosine, guanine, or thymine bases are linked by amide bonds. Peptidenucleic acids are synthesized by creating an amide bond between an aminogroup of the backbone and a carboxyl group of another peptide nucleicacid monomer.

The term “non-specific binding (NSB)” refers to a phenomenon where amacromolecule interacts with a surface and is typically dependent oncharge and/or hydrophobicity. In contrast, specific binding involvesselective interactions between an antigen and its correspondingantibody, or complementary strands of nucleic acids.

The term “LNA (Locked Nucleic Acid)” consist of conformationallyrestricted oligonucleotide analogs. LNA is a bicyclic nucleic acid wherea ribonucleoside is linked between the 2′-oxygen and 4′-carbon atomswith a methylene unit.

Analytes

The present disclosure provides articles and methods for detecting an“analyte” or a “target analyte”. Biological assays include at least onebiomolecule, which may take part in a biological assay as a bindingpartner (e.g., a receptor-ligand binding reaction, an antigen-antibodybinding reaction, an enzyme-substrate binding reaction, a hybridizationreaction between polynucleotide regions with at least partial homology).As will be appreciated by those in the art, a large number of analytesmay be used in the present disclosure. For example, any target analytecan be used which binds a bioactive agent or for which a binding partner(e.g. a drug candidate) is sought.

Suitable analytes include organic and inorganic molecules, includingbiomolecules. When detection of a target analyte is performed, suitabletarget analytes include, but are not limited to, an environmentalpollutant (including pesticides, insecticides, toxins, etc.); a chemical(including solvents, polymers, organic materials, etc.); therapeuticmolecules (including therapeutic and abused drugs, antibiotics, etc.);biomolecules (including hormones, cytokines, proteins, nucleic acids,lipids, carbohydrates, cellular membrane antigens and receptors (neural,hormonal, nutrient, and cell surface receptors) or their ligands, etc);whole cells (including prokaryotic (such as pathogenic bacteria) andeukaryotic cells, including mammalian tumor cells); viruses (includingretroviruses, herpes viruses, adenoviruses, lentiviruses, etc.); andspores; etc. Particularly preferred analytes are nucleic acids andproteins.

In a preferred embodiment, the target analyte is a protein. Proteindetection in microwell array assays is described, for example, in U.S.Pat. No. 6,942,968; U.S. Patent Application Publication Nos. US2003/0027327, US 2006/0228716, US 2006/0228722; and PCT InternationalPublication No. WO 03/016868; each of which is incorporated herein byreference in its entirety. As will be appreciated by those in the art,there are a large number of possible proteinaceous target analytes thatmay be detected or evaluated for binding partners using the articles ofthe present disclosure. Suitable protein target analytes include, butare not limited to, (1) immunoglobulins; (2) enzymes (and otherproteins); (3) hormones and cytokines (many of which serve as ligandsfor cellular receptors); and (4) other proteins.

Immunoglobulins include, but are not limited to, IgEs, lgGs and IgMs.Immunoglobulins include therapeutically or diagnostically relevantantibodies, including but not limited to, for example, antibodies tohuman albumin, apolipoproteins (including apolipoprotein E), humanchorionic gonadotropin, cortisol, α-fetoprotein, thyroxin, thyroidstimulating hormone (TSH), antithrombin, antibodies to pharmaceuticals(including antiepileptic drugs (phenytoin, primidone, carbariezepin,ethosuximide, valproic acid, and phenobarbital), cardioactive drugs(digoxin, lidocaine, procainamide, and disopyramide), bronchodilators(theophylline), antibiotics (chloramphenicol, sulfonamides),antidepressants, immunosuppresants, abused drugs (amphetamine,methamphetamine, cannabinoids, cocaine and opiates) and antibodies toany number of viruses (including orthomyxoviruses, (e.g. influenzavirus), paramyxoviruses (e.g. respiratory syncytial virus, mumps virus,measles virus), adenoviruses, rhinoviruses, coronaviruses, reoviruses,togaviruses (e.g. rubella virus), parvoviruses, poxviruses (e.g. variolavirus, vaccinia virus), enteroviruses (e.g. poliovirus, coxsackievirus),hepatitis viruses (including A, B and C), herpes viruses (e.g. Herpessimplex virus, varicella-zoster virus, cytomegalovirus, Epstein-Barrvirus), rotaviruses, Norwalk viruses, hantavirus, arenavirus,rhabdovirus (e.g. rabies virus), retroviruses (including HIV, HTLV-I and—II), papovaviruses (e.g. papillomavirus), polyomaviruses, andpicornaviruses, and the like), and bacteria (including a wide variety ofpathogenic and non-pathogenic prokaryotes of interest includingBacillus; Vibrio, e.g. V. cholerae; Escherichia, e.g. Enterotoxigenic E.coli, Shigella, e.g. S. dysenteriae; Salmonella, e.g. S. typhi;Mycobacterium e.g. M. tuberculosis, M. leprae; Clostridium, e.g. C.botulinum, C. tetani, C. difficile, C. perfringens; Cornyebacterium,e.g. C. diphtheriae; Streptococcus, S. pyogenes, S. pneumoniae;Staphylococcus, e.g. S. aureus; Haemophilus, e.g. H. influenzae;Neisseria, e.g. N. meningitidis, N. gonorrhoeae; Yersinia, e.g. Y.pestis, Pseudomonas, e.g. P. aeruginosa, P. putida; Chlamydia, e.g. C.trachomatis; Bordetella, e.g. B. pertussis; Treponema, e.g. T.palladium; and the like).

Enzymes (and other proteins) include, but not limited to, enzymes usedas indicators of or treatment for heart disease, including creatinekinase, lactate dehydrogenase, aspartate amino transferase, troponin T,myoglobin, fibrinogen, cholesterol, triglycerides, thrombin, tissueplasminogen activator (tPA); pancreatic disease indicators includingamylase, lipase, chymotrypsin and trypsin; liver function enzymes andproteins including cholinesterase, bilirubin, and alkaline phosphatase;aldolase, prostatic acid phosphatase, terminal deoxynucleotidyltransferase, and bacterial and viral enzymes such as HIV protease.

Hormones and cytokines (many of which serve as ligands for cellularreceptors) include, but are not limited to, erythropoietin (EPO),thrombopoietin (TPO), the interleukins (including IL-I through IL-17),insulin, insulin-like growth factors (including IGF-1 and -2), epidermalgrowth factor (EGF), transforming growth factors (including TGF-α andTGF-β), human growth hormone, transferrin, epidermal growth factor(EGF), low density lipoprotein, high density lipoprotein, leptin, VEGF,PDGF, ciliary neurotrophic factor, prolactin, adrenocorticotropichormone (ACTH), calcitonin, human chorionic gonadotropin, cotrisol,estradiol, follicle stimulating hormone (FSH), thyroid-stimulatinghormone (TSH), leutinzing hormone (LH), progeterone, testosterone.

Nonlimiting examples of other proteins include α-fetoprotein andcarcinoembryonic antigen (CEA).

In addition, any of the biomolecules for which antibodies may bedetected may be detected directly as well; that is, detection of virusor bacterial cells, therapeutic and abused drugs, etc., may be donedirectly.

In a preferred embodiment, the target analyte is a nucleic acid. Theseassays find use in a wide variety of applications. In a preferredembodiment, probes may be used in genetic diagnosis. For example, probescan be made to detect target sequences such as the gene for nonpolyposiscolon cancer, the BRCA1 breast cancer gene, P53, which is a geneassociated with a variety of cancers, the Apo E4 gene that indicates agreater risk of Alzheimer's disease, allowing for easy presymptomaticscreening of patients, mutations in the cystic fibrosis gene, cytochromep450s or any of the others well known in the art.

In an additional embodiment, viral and bacterial detection is done usingthe articles of the disclosure. In this embodiment, probes are designedto detect target sequences from a variety of bacteria and viruses. Forexample, current blood-screening techniques rely on the detection ofanti-HIV antibodies. The methods disclosed herein allow for directscreening of clinical samples to detect HIV nucleic acid sequences,particularly highly conserved HIV sequences. In addition, this allowsdirect monitoring of circulating virus within a patient as an improvedmethod of assessing the efficacy of anti-viral therapies. Similarly,viruses associated with leukemia, HTLV-I and HTLV-11, may be detected inthis way. Bacterial infections such as tuberculosis, chlamydia and othersexually transmitted diseases, may also be detected.

Flexible Microwell Arrays

FIG. 1 shows a top perspective exploded view of one embodiment of anarticle 100 of the present disclosure. The article 100 comprises amicrostructured layer 110 with an upper major surface 114 and a lowermajor surface 116. The upper major surface 114 comprises an array ofoptically-isolated microwells 122. The article 100 further comprises anoptically-transmissive flexible layer 130 coupled to the lower majorsurface 116 of the substrate 110. FIG. 1 further shows a set of axes toillustrate that, preferably, the microwells 122 are optically isolatedsuch that light is not substantially transmitted within the plane formedby the X-Y axes. However, light can be substantially transmitted fromthe microwells 122 in a direction that is predominantly oriented towardthe Z axis or, preferably, substantially parallel with the Z axis.

FIG. 4 shows a top perspective view of another embodiment of an article400 according to the present disclosure. In this embodiment, the article400 comprises a microstructured layer 410 with an upper major surface414 and a lower major surface 416. Microwells 422 extend below the uppermajor surface 414 of the microstructured layer 410. Also shown in FIG. 4is a tab portion 411 of the microstructured layer 410. The tab portion411 may be located in any position on the periphery of themicrostructured layer 410 and can serve as a region at which the article400 can be grasped and/or handled without contacting a microwell 422.The article may comprise a single tab region 411 or, preferably, aplurality of tab portions 411 (as shown) to provide multi-point controlof the article 400 during handling. Optionally, the microstructuredlayer 410 may comprise perforations 428, or the like, such that the tabportion 411 may be detached from the article 400 during or after use.

The microwells or chambers in the microstructured layer each have atleast one side wall, a bottom wall and both width and depth. Reactionmixtures, assay solutions, or microparticles can be deposited into eachmicrowell. The microwells are preferably of sufficient dimension andorder to allow for (i) the introduction of the necessary reactants intothe wells, (ii) chemical reactions or bioassays to take place within thewells and (iii) inhibition of mixing of particulate reactants and/oranalytes between wells. The microwell can be any shape. In oneembodiment, the shape of the microwell is preferably frustoconical, butthe microwell can be multi-sided so as to approximate a cylindrical orfrustoconical shape. The microwell can have a smooth wall surface. Insome embodiments, the microwell can have at least one irregular wallsurface. The bottom wall of the microwells can be either planar orconcave or convex.

In general, the microstructured layer permits optical detection of thecontents of each microwell. In some embodiments, the microstructuredlayer does not appreciably fluoresce. The microstructured layer ispreferably made of a material that facilitates detection of a chemicalreaction event or assay result in each microwell. For example, in atypical nucleic acid sequencing reaction, binding of a dNTP to a samplenucleic acid to be sequenced can be monitored by detection of photonsgenerated by enzyme reacting with phosphate moiety liberated in thesequencing reaction. Thus, having the base of the microwell and theoptically-transmissive flexible substrate be substantially transparentor light conductive facilitates detection of the photons.

In some embodiments, the microwells can be optically interrogatedthrough the opening of the microwells. In some embodiments, themicrowells can be optically interrogated through the bottom wall of themicrowells. In some embodiments, the microwells can be opticallyinterrogated through the opening of the microwells and through thebottom wall of the microwells.

In some embodiments, the microstructured layer comprises a colorant thatis substantially nontransmissive to selected wavelengths of light. Thedisclosed articles of the present disclosure provide for a microwellarray article wherein selected wavelengths of light are substantiallytransmitted through the bottom wall of each microwell while the selectedwavelengths of light are substantially absorbed by the side walls ofeach microwell, thereby reducing the incidence of optical cross-talkbetween adjacent microwells.

The microwells can be in a pattern, i.e. a regular design orconfiguration, or the microwells can be randomly distributed on thearray surface. In one embodiment, there is a regular pattern ofmicrowells such that the chambers may be addressed in the X-Y coordinateplane. “Pattern” in this sense includes a repeating unit, preferably onethat allows a high density of microwells on the substrate.

The shape of the microwells in the microstructured layer can be selectedto impart desirable effects on the signal production. That is, the wellscan be square, round or polygonal (e.g., pentagonal, hexagonal,octagonal) in shape. Preferably, the side walls of the microwells aresubstantially straight and form an angle (with the substantially planarsurface of the microwell array) that is greater than 90 degrees. Theangled side wall produces a frustoconical-shaped microwell in anembodiment wherein the opening and the bottom wall of the microwell arecircular in shape, for example.

The array pattern may comprise a composite array. As will be appreciatedby one skilled in the art, a configuration of a composite array is notlimited to the dimensions of a microtiter plate. A composite arrayconfiguration may be a single array, or may be a one-dimensionalcomposite of arrays, i.e. a composite array having only one array in afirst dimension and a plurality of arrays in a second dimension.Furthermore, a composite array can be a square, e.g., 2×2, 3×3, etc., orany other configuration, including, but not limited to, concentriccircles, spiral, rectangular, triangular, and the like. Preferably, thecomposite of arrays contain regularly spaced arrays in latticeconfiguration. In certain embodiments, the composite of arrays forms asquare or rectangular lattice.

In a preferred embodiment, the microwells are separated with spacesbetween each other. As is appreciated by those skilled in the relevantart, the spacing is determined by calculating the distance betweencenters. Varying the spacing between sites can result in the formationof arrays of high density, medium density or lower density. Themicrowells may be spaced any suitable distance apart. Spacing isdetermined by measuring the distance between the center points of twoadjoining microwells. The microwells are generally spaced between 5 μmand 200 μm apart. In some embodiments, the microwells may be spacedabout 10 μm to about 100 μm apart. In some embodiments, the microwellsmay be spaced about 12 μm to about 80 μm apart. In some embodiments, themicrowells may be spaced about 15 to about 50 μm apart. A particularadvantage of spacing wells apart is that commercial optical devices(e.g., cameras, scanners) can be used to analyze the arrays. Theresolution of optical devices may vary and arrays can be formed thatallow for detection on high or low resolution optical devices. In bothcases, various software packages (e.g., GENEPIX software package by AXONinstruments, ImagePro from Media Cybernetics, or others that areprovided with conventional fluorescent microscope scanning equipment)are used to analyze the image from the optical device. In a preferredembodiment, the software employs contrast-based or other imageprocessing algorithms to resolve the microwells and extract signalintensity information.

In some embodiments, a microparticle is disposed in at least onemicrowell of the microwell array. In some embodiments, a microparticleis disposed in at least 25% of the microwells of the microarray. In someembodiments, a microparticle is disposed in at least 50% of themicrowells of the microwell array. In some embodiments, a microparticleis disposed in at least 75% of the microwells of the microwell array. Insome embodiments, a microparticle is disposed in at least 90% of themicrowells of the microwell array. In some embodiments, a microparticleis disposed in about 100% of the microwells of the microwell array.

The size of the microwell can be made to accommodate any volume. In someembodiments, the microwell volume is about 1 to about 1000 picoliters.In some embodiments, the microwell volume is about 5 to about 100picoliters. In some embodiments, the microwell volume is about 20 toabout 50 picoliters. In some embodiments, the microwell volume is about25 picoliters.

The microwells may have any suitable width. In one embodiment, themicrowells have a diameter (width) in one dimension of about 3 μm toabout 100 μm. In one embodiment, the microwells have a diameter (width)in one dimension of about 5 μm to about 70 μm. In some embodiments, themicrowells have a diameter (width) in one dimension of about 10 μm toabout 50 μm.

The microwells may have any suitable depth. The depth of substantiallyall of the microwells is generally about 5 μm to about 100 μm. In someembodiments, the depth of substantially all of the microwells is about10 μm to about 60 μm. In some embodiments, the depth of substantiallyall of the microwells is about 30-40 μm. Substantially all of themicrowells means at least 90% of the microwells. In some embodiments,substantially all of the microwells means at least 95% of themicrowells. In some embodiments, substantially all of the microwellsmeans at least 97% of the microwells. In a further embodiment,substantially all of the microwells means at least 99%, more preferablyall, of the microwells. The depth of a microwell can be measured, forexample using a Wyko NT9100 Optical Profiler (Veeco, Plainview, N.Y.).Routine microwell depth measurements can be made using the areadifference plot feature of the instrument. The instrument can comparethe depth of a plurality of microwells to a reference point on a flatsurface of the substrate to provide an average microwell depth.

The array preferably comprises a sufficient number of microwells tocarry out such numerous individual assays. The array can contain anynumber of microwells. Depending on the end use of the array, substratesare made to comprise a certain number of microwells (e.g., greater than20,000 microwells, greater than 100,000 microwells, greater than1,000,000 microwells, greater than 5,000,000 microwells).

FIG. 2A shows a cross-sectional view of one embodiment of a microwellarray article 200 according to the present disclosure. The article 200comprises a microstructured layer 210. The microstructured layer 210comprises an array of optically isolated microwells 222. Each microwell222 comprises an opening 224, a bottom wall 228, and at least one sidewall 226 extending from the opening 224 to the bottom wall 228.

The microstructured layer 210 can be formed from a suitable materialusing a curing process described herein. Suitable materials includepolymer materials (e.g., acrylic-based resins derived from epoxies,polyesters, polyethers, and urethanes; ethylenically unsaturatedcompounds; aminoplast derivatives having at least one pendant acrylategroup; polyurethanes (polyureas) derived from an isocyanate and a polyol(or polyamine); isocyanate derivatives having at least one pendantacrylate group; epoxy resins other than acrylated epoxies; and mixturesand combinations thereof) that can be processed to form a unitary,flexible microstructured layer 210 comprising a plurality of microwells222, each microwell 222 with a bottom wall 228 that is, for example,about 0.2 μm to about 6 μm thick. In some embodiments, the polymermaterials are substantially impermeable to water. In some embodiments,the substrate can be coated with one or more materials, as describedherein.

The microstructured layer 210 is coupled to an optically-transmissiveflexible layer 230. In some embodiments, the optically-transmissiveflexible layer 230 substantially permits the transmission of visiblewavelengths of light there through. In some embodiments, theoptically-transmissive flexible layer 230 further permits thetransmission of ultraviolet wavelengths of light there through. In someembodiments, the optically-transmissive flexible layer 230 comprises apolymeric material. Nonlimiting examples of suitable polymeric materialsinclude polyethylene naphthalate (PEN), PET (polyethylene terephthalate,PEN (polyethylene naphthalate), HDPE (high density polyethylene, LDPE(low density polyethylene), LLDPE (linear low density polyethylene). PETand PEN are particularly preferred.

Optionally, the microwell array article 200 can comprise an adhesivelayer 240. In some embodiments, the adhesive layer 240 can beincorporated into the microwell array article 200 by coating an adhesiveonto the optically-transmissive flexible layer 230. In some embodiments,the adhesive layer 240 can be transferred to the microwell array article200 by transferring the adhesive layer 240 from a carrier to theoptically-transmissive flexible layer 230 via lamination processes thatare known in the art, for example. Preferably, the adhesive layer 240substantially permits the transmission of light (e.g., ultravioletand/or visible wavelengths of light).

The optional adhesive layer 240 can couple the microwell array article200 to a variety of substrates 250 that can serve at least one of avariety of functions. In some embodiments, substrate 250 can be aflexible carrier (e.g., paper, coated paper, polymeric film, metal film)that functions to carry the microstructured layer 210 or the materialsthat form the microstructured layer 210 during a processing step. Insome embodiments, the substrate 250 can be a rigid or a flexiblematerial (e.g., a glass slide, a plastic film, coated paper) and canfunction as a protective layer to retain functional properties (e.g.,structure, shape, size, chemical integrity, optical properties, and/oradhesion properties) associated with the article. In some embodiments,the substrate 250 may be a component of a package to contain themicrowell array article 200, as described herein and in U.S. PatentApplication No. 61/236,612, filed Nov. 23, 2009 and entitled “CARRIERFOR FLEXIBLE MICROASSAY DEVICE AND METHODS OF USE”, which isincorporated herein by reference in its entirety. In some embodiments,the substrate 250 may be a component of an imaging system (e.g., acamera, a lens, a fiber optic bundle).

The substrate 250 can be a flexible component that can be used for avariety of purposes. Nonlimiting examples of flexible substrates includepolymer films, metal films, or paper. In some embodiments, the substrate250 is a carrier (e.g., a release liner) that is coated with adhesive240 in order to transfer the adhesive 240 to the optically-transmissiveflexible layer 230. Preferably, in these and other embodiments, thesubstrate 250 is coated with a release chemistry such as a silicone,fluorosilicone, wax, or other low surface energy material to facilitaterelease of the adhesive layer 240 from the substrate 250. Flexiblesubstrates can be used for processing, carrying, and/or protecting themicrowell array articles from damage or contamination.

The substrate 250 can be a rigid structural component (e.g., a camera, afiber optic faceplate, a microscope slide, a mirror) that causes amicrowell array article to be inflexible or to retain structural memory.By coupling the microwell array article to a rigid substrate, thearticle can retain a shape that is optically interrogatable. The step ofcoupling the microwell array article to a rigid substrate is carried outby contacting the article directly to the substrate or by coating eitherthe article and/or the rigid substrate with a bonding agent and thencontacting the article/bonding agent to the substrate orsubstrate/bonding agent to the article. The result of the coupling stepwill be to cause the microwell array article to be attached to a rigidsubstrate, preferably such that it does not rotate, bubble, warp, curl,tear, or otherwise deform in a manner which adversely influences theability to optically interrogate the microwells or which adverselyinfluences the flow of fluids over the article and selected microwelllocations therein.

A bonding agent useful in the adhering step of the method of thedisclosure can be any substance that is capable of securing theattachment of the microwell array article to the substrate withoutadversely influencing the ability to optically interrogate themicrowells and which does not cause the adverse degradation of thesubstrate or the microwell array article. As will be appreciated by oneskilled in the art, when the article is coated with the bonding agent,the back surface of the article will be coated; that is, the surfacecoated with the bonding agent is the surface of the article notcontaining the formed features such as microwells. Suitable bondingagents include, but are not limited to, liquid epoxies; glues oradhesives. Preferably, a pressure sensitive adhesive is used.

A rigid substrate will either substantially prevent the microwell arrayarticle from deforming or will, upon deformation of the article, causethe article to substantially return to the article's intended shape. Aswill be recognized by one skilled in the art, “substantially prevent”,“substantially return”, and like terms refer to structural propertieswhere the shape of the structure is maintained or reinstated in such away as to permit the desired optical interrogation of the microwellarrays, in accordance with the methods taught and cited herein. In someembodiments, a rigid substrate is flat or planar such that substantiallyall microwells of an array can be accurately and simultaneouslydetected. An exemplary rigid substrate is a glass slide. In addition,the rigid substrate can serve to keep the article in a planar or flatconfiguration. As is known in the art, many detection techniques, forexample, fluorescence detection, rely on very shallow depth of fielddetection methods using, for example, CCD cameras and confocalmicroscopes. Since many flexible articles cannot be made sufficientlyflat or planar for such detection methods, a rigid substrate is used tomaintain the article in a flat or planar configuration.

A rigid substrate can be formed from any of a variety of materials andwill be selected according to the desired properties of the rigidsubstrate, including, but not limited to the above-discussed structuralproperties and other structural properties such as flatness, strength,stiffness, thickness, low thermal expansion coefficient, opticalproperties and chemical properties such as microstructured layercompatibility. For example, a rigid structure can be selected to haveoptical properties that include, but are not limited to having lowautofluorescence, or being transparent, selectively transparent, havinga selected refractive index, absorptive, selectively absorptive, opaqueor reflective. In addition, a metal or metal-coated rigid structure canbe employed to enhance signal collection from the arrays.

Compositions for a rigid substrate which demonstrate the aboveproperties include metals, such as aluminum, iron, steel, variousalloys, and the like; ceramics; composites such as fiberglass; siliconor other semiconductor materials; glass; rigid plastics or polymers; andthe like.

FIG. 2B is an enlarged view of one of the microwells 222 of themicrowell array article 200 of FIG. 2A. FIG. 2B shows themicrostructured layer 210 comprising microwells 222 with a bottom wall228. Also shown are the optically-transmissive flexible layer 230,optional adhesive layer 240 and optional substrate 250. The “t”indicates the minimum distance a signal must cross in order to bereceived by an optical device (not shown) coupled to the bottom surfaceof the microwell array article 200. In order for an optical signal to bedetected by an optical device coupled to the bottom or the microwellarray article 200, the signal must pass through the bottom wall 228, theoptically-transmissive flexible layer 230, and the adhesive layer 240,if present. Thus, each of the bottom wall 228, theoptically-transmissive flexible layer, and the adhesive layer 240, ifpresent, must be transmissive to the wavelength or wavelengths of lightto be detected.

Once made, the articles of the disclosure find use in a number ofapplications. In a preferred embodiment, the articles are used to probea sample solution for the presence or absence of a target sequence,including the quantitation of the amount of target sequence present. Inanother preferred embodiment, the articles are used to determine anucleic acid sequence.

The articles of the disclosure are useful for any of a variety ofnucleic acid assays including SNP identification, sequencing,amplification, hybridization, genotyping, nucleic acid quantitation, andthe like. Methods for carrying out such assays are taught in, forexample, U.S. Patent Application Publication No. US 2006/0228722 and PCTInternational Publication No. WO 03/016868, each of which is hereinincorporated by reference in its entirety.

Process for Making Flexible Microwell Arrays

The present disclosure provides a process for making flexible microwellarray articles. The process comprises casting a curable resincomposition onto a microstructured tool, curing the resin composition,and removing the resultant article from the tool. Similar processes aredescribed in U.S. Pat. Nos. 5,175,030; 5,183,597; 5,384,571; 5,691,846;and 6,778,336; and in PCT Publication No. WO 9511464, each of which isincorporated herein by reference in its entirety. Briefly summarizing,the process for making such microwell array articles comprises the stepsof:

-   -   a) providing        -   a tool having a molding surface with a plurality of            projections extending therefrom suitable for forming the            microstructure elements (e.g., microwells);        -   a flowable, curable resin composition; and        -   an optically-transmissive flexible layer having first and            second major surfaces, wherein the first major surface of            the optically-transmissive flexible layer is surface-treated            to promote adhesion to a cured resin composition, wherein            the thickness of the optically-transmissive flexible layer            is about 50 μm or less;    -   b) applying to the molding surface a volume of the resin        composition suitable for forming the desired microstructure        elements;    -   c) contacting the resin composition with the first major surface        of the optically-transmissive flexible layer;    -   d) curing the resin composition while in contact with the        flexible layer to form a microwell array article comprising a        cured microstructured layer bonded to the flexible layer, and    -   e) removing the microwell array article from the tool.

The tool should be such that the projections will not deform undesirablyduring fabrication of the microwell array article, and such that themicrowell array can be separated therefrom after curing. Illustrativeexamples of substrates known to be useful for forming tools forreplication of microwell array articles include materials that can bedirectly machined. Such materials preferably machine cleanly withoutburr formation, exhibit low ductility and low graininess, and maintaindimensional accuracy after formation of the projections. A variety ofmachinable plastics (including both thermoset and thermoplasticmaterials), e.g., acrylics, and machinable metals, preferablynonferrous, e.g., aluminum, brass, copper, and nickel are known. In manyinstances, it may be desired to use a first or later generationreplicate of a machined or shaped surface as the tool (i.e., the memberon which the disclosed microwell arrays are formed). Depending upon thetool used and the nature of the resin composition, the cured microwellarray may separate from the tool readily or a parting layer may benecessary to achieve desired separation characteristics. Illustrativeexamples of parting layer materials include an induced surface oxidationlayer, an intermediate thin metallic coating, chemical silvering, and/orcombinations of different materials or coatings that create a low energysurface, such as silicones or fluorinated materials, for example. Ifdesired, suitable agents may be incorporated into the resin compositionto achieve desired separation characteristics.

As discussed above, the tool can be made from polymeric, metallic,composite, or ceramic materials. In some embodiments, curing of theresin will be performed by applying radiation through the tool. In suchinstances, the tool should be sufficiently transparent to permitirradiation of the resin there through. Illustrative examples ofmaterials from which tools for such embodiments can be made includepolyimide, polyacrylate, polyolefin, polycarbonates, and cured urethaneacrylates. Metal tools are typically preferred, however, as they can beformed in desired shapes, are durable, and also can provide excellentoptical surfaces in the substrate.

A flowable resin is applied to the molding surface of the tool. Theresin should be such that it flows, optionally with applied vacuum,pressure, or mechanical means, into areas and/or cavities in the moldingsurface. It is preferably applied in sufficient quantity that it atleast substantially fills the cavities and/or surrounds projections onthe molding surface.

The method of the disclosure also includes a step of releasing themicrowell arrays from the surface of the template structure. Generally,the step of removing the moldable material from the surface of thetemplate structure will be carried out in such a way as to permit themoldable material to maintain a shape that is fully complementary to thetemplate structure, and thus, the moldable material is removed from thesurface of the template structure in the form of the microstructuredlayer.

Although the microwell array articles are characterized as havingsubstantially unchanged features, it will be understood that thearticles are not required to be rigid or retain structural memory beyondthat of maintaining the shape of the features. The flexible microwellarray articles can be stored in a compact form, such as in rolled formon a spool, as stacked sheets, or any other configuration for convenientstorage.

FIG. 5 shows an exemplary apparatus for making microwell array articlesaccording to the present disclosure. An optically-transmissible flexiblelayer 530 (e.g., a polymeric film) is threaded from an unwind idler 581,over a first nip roller 587, around a portion of a patterned tool roll583 comprising microstructured projections 584, around a portion of asecond nip roller 588, and onto a rewind idler 582. A resin composition586 is cast from a resin hopper 585 directly onto the opticallytransmissible flexible layer 530 at a location proximate a patternedtool roll 583. The resin/flexible layer combination is then contactedwith the patterned tool roll 583 with pressure being applied throughappropriate setting (described below) of first nip roller 587. Pressureapplied to the first nip roller 587 serves to control the amount ofresin extending between the microstructured projections 584 of patternedtool roll 583 and the optically-transmissive flexible layer 530,allowing control of the thickness of the bottom wall of the microwellsformed by the microstructured projections 584. The resin composition 586is cured by exposure to actinic radiation from a first radiation source541, which may include a plurality of radiation-emitting bulbs, forexample. The cured microwell array article 500 is pulled out of thepatterned tool roll 583 at second nip roller 588 and collected onto therewind idler 582. In some embodiments, an optional second radiationsource 592 is positioned to direct radiation onto the microstructuredside of the microwell array article 500 to complete the curing process.

In some embodiments, the patterned tool roll 583 may be heated tomodulate the viscosity of the resin composition 586, thereby providingan additional means to control the thickness of the bottom wall.

In choosing the polymeric components of composite microarray materialsof the present disclosure, it is important to select compatiblepolymeric materials for the microstructured layer and flexible layer. Apreferred aspect of compatibility is that the material of the resincomposition of the microstructured layer be capable of bonding to theoptically-transmissive flexible layer when cured. In certain preferredembodiments, a major surface of the optically-transmissive flexiblelayer is surface-treated to promote bonding with the cured polymer thatforms the microstructured layer. Suitable surface treatments include,for example, radiation treatments, corona discharge treatment (e.g., airor nitrogen corona discharge), flame treatment, plasma treatment, highenergy UV treatment (e.g., flashlamp treatments), and chemical primingtreatment (e.g. chemical reactive coatings).

Resins selected for use in the microarray of articles preferably yieldresultant products that provide highly efficient transmission of lightto a detection device or system, as well as sufficient durability andchemical stability. Illustrative examples of suitable polymers include aresin selected from the group consisting of acrylic-based resins derivedfrom epoxies, polyesters, polyethers, and urethanes; ethylenicallyunsaturated compounds; aminoplast derivatives having at least onependant acrylate group; polyurethanes (polyureas) derived from anisocyanate and a polyol (or polyamine); isocyanate derivatives having atleast one pendant acrylate group; epoxy resins other than acrylatedepoxies; and mixtures and combinations thereof. Polymers such aspoly(carbonate), poly (methylmethacrylate), polyethylene terephthalate,aliphatic, polyurethane, and cross-linked acrylate such as mono- ormulti-functional acrylates or acrylated epoxies, acrylated polyesters,and acrylated urethanes blended with mono- and multi-functional monomersare typically preferred.

These polymers are typically preferred for one or more of the followingreasons: high thermal stability, environmental stability, and clarity,excellent release from the tooling or mold and high receptivity forreceiving a coating.

Other illustrative examples of materials suitable for forming themicroarray elements are reactive resin systems capable of beingcross-linked by a free radical polymerization mechanism by exposure toactinic radiation, for example, electron beam, ultraviolet light, orvisible light. Radiation-initiated cationically polymerizable resinsalso may be used. Reactive resins suitable for forming the microarrayelements may be blends of photoinitiator and at least one compoundbearing an acrylate group. Preferably the resin blend contains amonofunctional, a difunctional, or a polyfunctional compound to ensureformation of a cross-linked polymeric network upon irradiation.Chemical-mediated polymerizable resins may be used (e.g., these may bepolymerized by thermal means with the addition of a thermal initiatorsuch as benzoyl peroxide). U.S. Pat. Nos. 6,395,124 and 6,692,611, eachof which is incorporated herein by reference in its entirety, discloseexemplary photoinitiators that are suitable for free radical initiationof polymerization using wavelengths of light that are in the visiblerange (e.g., longer than 400 nm).

Illustrative examples of resins that are capable of being polymerized bya free radical mechanism that can be used herein include acrylic-basedresins derived from epoxies, polyesters, polyethers, and urethanes,ethylenically unsaturated compounds, aminoplast derivatives having atleast one pendant acrylate group, isocyanate derivatives having at leastone pendant acrylate group, epoxy resins other than acrylated epoxies,and mixtures and combinations thereof. The term acrylate is used here toencompass both acrylates and methacrylates. U.S. Pat. No. 4,576,850,which is incorporated herein by reference in its entirety, disclosesexamples of crosslinked resins that may be used in microwell arrays ofthe present disclosure.

Ethylenically unsaturated resins include both monomeric and polymericcompounds that contain atoms of carbon, hydrogen and oxygen, andoptionally nitrogen, sulfur, and halogens may be used herein. Oxygen ornitrogen atoms, or both, are generally present in ether, ester,urethane, amide, and urea groups. Ethylenically unsaturated compoundspreferably have a molecular weight of less than about 4,000 andpreferably are esters made from the reaction of compounds filmscontaining aliphatic monohydroxy groups, aliphatic polyhydroxy groups,and unsaturated carboxylic acids, such as acrylic acid, methacrylicacid, itaconic acid, crotonic acid, iso-crotonic acid, maleic acid, andthe like. Such materials are typically readily available commerciallyand can be readily cross linked.

Some illustrative examples of compounds having an acrylic or methacrylicgroup that are suitable for use in the disclosure are listed below:

-   -   (1) Monofunctional compounds: ethylacrylate, n-butylacrylate,        isobutylacrylate, 2-ethylhexylacrylate, n-hexylacrylate,        n-octylacrylate, isooctyl acrylate, bornyl acrylate,        tetrahydrofurfuryl acrylate, 2-phenoxyethyl acrylate, and        N,N-dimethylacrylamide;    -   (2) Difunctional compounds: 1,4-butanediol diacrylate,        1,6-hexanediol diacrylate, neopentylglycol diacrylate, ethylene        glycol diacrylate triethyleneglycol diacrylate, tetraethylene        glycol diacrylate, and diethylene glycol diacrylate; and    -   (3) Polyfunctional compounds: trimethylolpropane triacrylate,        glyceroltriacrylate, pentaerythritol triacrylate,        pentaerythritol tetraacrylate, and        tris(2-acryloyloxyethyl)isocyanurate.        Preferably, blends of mono-, di-, and polyfunctional acrylate        containing materials are used. One skilled in the art will        understand that varying the ratios among these components will        determine the mechanical properties of the fully cured material.

Some representative examples of other ethylenically unsaturatedcompounds and resins include styrene, divinylbenzene, vinyl toluene,N-vinyl formamide, N-vinyl pyrrolidone, N-vinyl caprolactam, monoallyl,polyallyl, and polymethallyl esters such as diallyl phthalate anddiallyl adipate, and amides of carboxylic acids such asN,N-diallyladipamide.

Illustrative examples of photopolymerization initiators that can beblended with acrylic compounds in microwell array articles of thepresent disclosure include the following: benzil, methyl o-benzoate,benzoin, benzoin ethyl ether, benzoin isopropyl ether, benzoin isobutylether, etc., benzophenone/tertiary amine acetophenones such as2,2-diethoxyacetophenone, benzyl methyl ketal, 1-hydroxycyclohexylphenylketone, 2-hydroxy-2-methyl-1-phenylpropan-1-one,1-(4-isopropylphenyl)-2-hydroxy-2-methylpropan-1-one,2-benzyl-2-N,N-dimethylamino-1-(4-morpholinophenyl)-1-butanone,2,4,6-trimethylbenzoyl-diphenylphosphine oxide,phenyl-bis(2,4,6-trimethylbenzoyl)phosphine oxide,2,4,6-trimethylbenzoylphenyl phosphinate, 2-methyl-1-4(methylthio),phenyl-2-morpholino-1-propanone, bis(2,6-dimethoxybenzoyl)(2,4,4-trimethylpentyl)phosphine oxide, etc. The compounds may be usedindividually or in combination.

Cationically polymerizable materials including but are not limited tomaterials containing epoxy and vinyl ether functional groups may be usedherein. These systems are photoinitiated by onium salt initiators, suchas triarylsulfonium, and diaryliodonium salts.

Preferably, the optically-transmissive flexible layer used in the methodof the present disclosure is a polymeric material selected from thegroup consisting of PET (polyethylene terephthalate, PEN (polyethylenenaphthalate), HDPE (high density polyethylene, LDPE (low densitypolyethylene), LLDPE (linear low density polyethylene). PET and PEN areparticularly preferred, other light transmissive elastomer, andcombinations thereof. Such materials typically impart desireddurability, flexibility, and light transmissivity to the resultantmicrowell array articles, while permitting desired bonding with themicrostructured layer.

The optically-transmissive flexible layer preferably comprises a polymerhaving a glass transition temperature greater than about 70° C. and amodulus about 3×10⁹ Pa or greater. The polymer preferably is such thatthe optically-transmissive flexible layer retains its physical integrityunder the conditions it is exposed to as the resultant microwell arrayis formed. Preferred polymeric materials used in theoptically-transmissive flexible layer are resistant to degradation by UVlight radiation so that the microarray articles can be used forapplications involving fluorescence-based detection. Theoptically-transmissive flexible layer should be light transmissive andpreferably is substantially transparent. For instance, films with amatte finish that become transparent when the resin composition isapplied thereto, or that only become transparent during the fabricationprocess, e.g., in response to the curing conditions, are useful herein.

The optically-transmissive flexible layer may be either a single layeror multi-layer component as desired. If multilayer, the layer to whichthe microstructured layer is bonded should have the properties describedherein as useful in that regard with other layers not in contact withthe microstructured layer having selected characteristics (e.g.,antireflective, optical transmissivity) as necessary to impart desiredcharacteristics to the resultant microwell array. Advantageously,multilayer films may impart significant structural integrity (e.g.,tear-resistance) to the microwell array articles. Further, the filmsused in a multi-layer construction can be selected to transmit and/orreflect selected wavelengths of light.

The optically-transmissible flexible layer is preferably about 2 micronsto about 50 microns thick. More preferably, the optically-transmissiveflexible layer is about 4 microns to about 25 microns thick. Even morepreferably, the optically-transmissive flexible layer is about 2 micronsto about 12 microns thick. Preferred materials for theoptically-transmissive flexible layer include polyethylene naphthalate(PEN) and polyethylene terephthalate (PET). Preferably, theoptically-transmissive flexible layer is available in roll form, to beused in a process as shown in FIG. 5.

Colorants, processing aids such as antiblocking agents, releasingagents, lubricants, and other additives may be added to one or both ofthe microstructured layer and optically-transmissive flexible layer ifdesired. The colorant may comprise a dye that is dissolved in the resincomposition from which the microstructured layer is formed.Alternatively, or additionally, the colorant may comprise a pigment thatis uniformly dispersed in the resin from which the microstructured layeris formed. The particular colorant selected depends on the desiredtransmissivity (and/or nontransmissivity) for particular colors oflight; colorants typically are added at about 0.01 to 5.0 weightpercent. Preferred colorants do not substantially interfere withreactants or reactions that are conducted in the microwells.

The amount of a colorant added to the resin from which themicrostructured layer or in a coating that is applied to themicrostructured layer can be adjusted depending upon one or more of thefollowing parameters: the light-absorbing properties of the colorant,the distance between the closest adjacent microwells, and the thicknessof the bottom wall of the microwells. It will be recognized by a personof ordinary skill in the relevant art that, as the concentration of thecolorant increases, the amount of light absorbed by the microstructuredlayer will increase.

Microstructured layers containing high concentrations of colorant willabsorb relatively more light and, thus, will permit relatively closerspacing of the microwells. In these embodiments, however, the bottomwall should be proportioned relatively thinner, in order to permit thetransmission of substantially all of the light from the microwellthrough the bottom wall.

In contrast, microstructured layers containing lower concentrations ofcolorant will absorb relatively less light and, thus, will requirerelatively greater spacing of the microwells in order to prevent lightfrom passing laterally through the microstructured layer from onemicrowell to an adjacent microwell. In these embodiments, however, thebottom wall may be proportioned relatively thicker and still permit thetransmission of substantially all of the light from the microwellthrough the bottom wall.

In some embodiments, prior to the step of contacting the molding surfaceof the tool with a flowable material, a releasing agent is applied tothe surface of the tool. As used herein a “releasing agent” is anycompound, composition, or other substance, which aids in the separationof the moldable material from the surface of the tool in forming anarticle. Useful releasing agents include silicone oil, polyvinylalcohol, fluorinated silane or other known releasing agent. Selection ofthe type and amount of a releasing agent will depend on several easilydeterminable factors such as compatibility with reactions that areconducted in the resultant microwell array, the strength of the tool,the curable resin composition, the environmental conditions of thecontacting and molding process, the degree of tolerance for distortionsor imperfections in the article, and the like.

In some embodiments, in addition to the steps described above, theprocess for making a microwell array article further comprises a step toremove (e.g., by ablation or etching) a portion of the microstructuredlayer and/or the optically-transmissive flexible layer. In theseembodiments, the microwell array article is subjected to a process thatselectively removes material from at least one surface (e.g., the uppersurface, the lower surface, or the upper and lower surfaces) of themicrowell array article.

Thus, in some embodiments, wherein the ablation or etching step isapplied only to the upper surface of the article, the relatively thinbottom wall of each microwell can be selectively removed from thearticle, thereby causing the optically-transmissive flexible layer toform the bottom of each microwell in the array. Advantageously, thisprocess can provide for better recovery of light transmitted from theinterior of individual microwells, especially when high concentrationsof colorant are used.

In alternative embodiments, wherein the ablation or etching step isapplied only to the lower surface of the article, a portion of arelatively thick optically-transmissive flexible layer can be uniformlyremoved to produce a thinner optically-transmissive flexible layer.Advantageously, this process can provide for less optical cross-talk, asshown in FIGS. 3A and 3B and described herein.

When both the upper and lower surfaces of the article are subjected tothe ablation process, the resultant microwell array articles can haveincreased light transmission from the microwells and decreased opticalcross-talk.

Processes for controllably ablating thin layers of flexible polymericfilms are known in the art and include, for example, plasma treatment,reactive ion etching, laser ablation, or chemical etching (e.g., usinghydrolytic agents, such as a solution containing 40% w/v potassiumhydroxide and 20% w/v ethanolamine to chemically etch PET film).

Thin Film Coatings

The surface of the array substrate may be coated with a thin layer ofmaterial to enhance the properties and functions of the microwells. Thecoating may protect the contents of the solution in the reaction chamberfrom the deleterious effects of the array substrate, withoutcompromising the utility or ease of fabrication of the array. Thecoating may also provide a uniform surface composition allowing foruniform modification of the reaction chamber surface (e.g., with amonolayer).

The present disclosure provides for the application of a coating to thearray substrate. Such coatings are designed to improve the propertiesand functions of the array, including compatibility of the reactionmixture or assay solution. The coating may provide a barrier between thesolution contained in the microwell and the substrate, and prevents bothleaching of the substrate material into the solution and contact of thecontents in the microwell with the substrate.

In some embodiments, the substrate is coated with a thin film comprisedof a material typically known to be compatible with components found inassay solutions and chemical reaction mixtures. In one embodiment, thecoating may be impermeable to water. In another embodiment, the coatingcan provide for a uniform surface composition. Preferably, the coatingis optically transparent and such transparency facilitates detection.

Other desirable properties of the coating include durability,compatibility with the substrate materials, well-understood depositionparameters, and resistance to relatively high temperatures. In oneembodiment, the coating is adhesive to glassy materials. The coating maypreferably minimize non-specific absorption of macromolecules to theside and bottom walls of the microwells. In one embodiment, the coatingallows for easy attachment of reactants (e.g. proteins and nucleicacids) and does not negatively affect the activity of immobilizedreactants, but rather in some instances, can increase their stability.

The coating may be deposited on the surface of the array (i.e., the arealying outside the microwells), on the bottom walls of a microwell,and/or on the side walls of a microwell. In one embodiment, the thinfilm is deposited on the entire substrate. In another embodiment, thecoating is deposited on the surface of the array. In a furtherembodiment, the coating is deposited on the bottom wall of eachmicrowell. In a further embodiment, the coating is deposited on the sidewalls of each microwell. In some embodiments, the coating is depositedon the bottom wall and side walls of each microwell and on the surfaceof the substrate.

The term “coating” refers to a relatively thin composition (e.g., afilm) with a thickness that is significantly smaller than othercharacteristic dimensions of the substrate. In a preferred embodiment,the coating is uniform and conformal to the microstructured layer, witha thickness of about 25 to about 1000 nanometers. The thickness of thecoating may be non-uniform over the surface of the array. For example,in one embodiment, the thickness of the thin film coating can be about50-500 nm on the top surface of the array substrate; about 25-250 nm onthe side walls of the microwells, and about 50-500 nm on the bottomwalls of the microwells.

Many different types of materials can be used as a coating. Thecomposition of a coating material will depend on the array substrate,the application, and the method of coating deposition. In oneembodiment, a coating is a polymer (e.g., an inorganic polymer). Acoating can be a non-metal oxide (e.g. silicon dioxide (SiO₂)). Othercoatings may be, for example, a metal alloy, a metal or semi-conductoroxide, nitride, carbide or boride. Other materials for coating thesubstrate may include gold layers (e.g. 24 karat gold). Many coatingsare commercially available.

Coating materials also include those systems used to attach apolypeptide or polynucleotide to a substrate. Organosilane reagents,which allow for direct covalent coupling of proteins via amino,sulfhydryl or carboxyl groups, can be used to coat the array substrate.Additional coating substances include photoreactive linkers (e.g.photobiotin).

Other coating materials include polymeric materials such as hydrophilicpolymer gels (e.g., polyacrylamide and polysaccharides), which may bepolymerized directly on the surface of the substrate or polymer chainsthat are covalently attached to the substrate. Other coating materialsalso include passively-adsorbed layers (e.g., biotin-binding proteins).The substrate can also be coated with an epoxide which allows thecoupling of reagents via an amine linkage.

In a preferred embodiment, the coating is SiO₂. The substrate can becoated with a film coating of SiO₂. Film coatings of SiO₂ are opticallytransparent, function efficiently as a water barrier in thicknesses downto 10 nm, adhere to glassy materials, and withstand harsh cleaningprocedures and relatively high temperatures. Further, the surfaceproperties of SiO₂ are well known, as are methods for modifying theseproperties. Further, SiO₂ has been shown to be compatible withmicroscale biological assays such as polymerase chain reaction (“PCR”),for example.

Coated microwells can be biologically or chemically functionalized. Anyof the film materials discussed can be derivatized with one or morefunctional groups, commonly known in the art for the immobilization ofpolypeptides and/or polynucleotides, e.g. metal chelating groups (e.g.nitrilo, triacetic acid, iminodiacetic acid, pentadentate chelator). Inone embodiment, the coated microwell is modified to contain functionalgroups that can be used to attach or capture, either covalently ornon-covalently, reactants or analytes to the coated walls of themicrowell. “Chemically-functionalized microwells” in this contextinclude, but are not limited to, the addition of functional groupsincluding amino groups, carboxy groups, oxo groups and thiol groups,that can be attached to the coated surface of the microwell and be usedto attach or capture reactants or analytes on the same surface.Biological modifications of the coated microwell include, for example,the attachment of binding ligands or binding partner pairs, includingbut not limited to, antigen/antibody pairs, enzyme/substrate orenzyme/inhibitor pairs, receptor-ligand pairs, carbohydrates and theirbinding partners (lectins, etc.).

Method of Preparing a Flexible Microwell Array for Microanalysis

The present disclosure provides a method to prepare a flexible microwellarray article for microanalysis. The method comprises providing anarticle that includes first and second protective layers with a flexiblemicrowell array comprising an adhesive layer disposed there between. Themethod further comprises providing a component of an optical system. Themethod further comprises pulling the second protective layer away fromthe microwell array such that the second protective layer is detachedfrom the microwell array and/or the first protective layer. The methodfurther comprises applying the lower major surface of the microwellarray to the optical system component.

FIG. 8A shows one embodiment of a microwell article comprisingprotective layers. The article 800 comprises a flexible microwell array810 that includes an adhesive layer 815 on the major surface of themicrowell array article 810 opposite the major surface that includes themicrowells (microwells are not shown in FIGS. 8A-8F). A first protectivelayer 820 is coupled to the microwell array 810 on the major surfaceopposite the adhesive layer 815. A shielding element 840 is coupled tothe adhesive layer 815. A second protective layer 830 is coupled to theshielding element 840. FIG. 8A shows the body region (“a”), which issubstantially coextensive with the microwell array 810, of the first andsecond protective layers (820 and 830, respectively). Extending beyondone edge of the microwell array 810 is the tab region (“b”) of the firstand second protective layers (820 and 830, respectively). Extendingbeyond another edge of the microwell array 810 is a margin area (“c”).The margin area “c”, is a portion of the article outside of the tabregion where the peripheral boundaries of the first and secondprotective layers overlap and extend beyond the peripheral boundary ofthe microwell array.

FIG. 8B shows a top view of the article of FIG. 8B. FIG. 8B illustratesthat the first protective layer body region 820 a of the article 800 issubstantially coextensive with the microwell array 810, thereby forminga covering over the microwells 812. The first protective layer tabregion 820 b extends from a portion of the first protective layer bodyregion 820 a. Like the tab region 820 b, the first protective layermargin area 820 c extends from the first protective layer body region820 a beyond the peripheral boundary of the microwell array 810. Alsoshown in FIG. 8B are alignment indicia 850. Alignment indicia can be anymarking or combination of markings (e.g., lines, dots, lettering,symbols, or the like) that can serve as a point of reference to properlyalign the microwell array 815 with a component of an optical system(e.g., a camera, a fiber optic array, a line scanner (not shown)).

FIGS. 9A-9F illustrate one embodiment of the method to prepare aflexible microwell array for microanalysis. FIG. 9A shows an article 900comprising a flexible microwell array 910 according to the article ofFIG. 9A. The microwell array 910 comprises an adhesive layer 915. Thearticle further comprises a shielding element 940 releasably bonded tothe adhesive layer 915, a first protective layer 920 releasably bondedto the microwell array 910, and a second protective layer 930 releasablybonded to the shielding element 940. In this step, the first protectivelayer 920 and second protective layer 930 are grasped and generallypulled in opposing directions to separate the layers.

FIG. 9B shows the two components (I and II) resulting from theseparation of the protective layers during the step described in FIG.9A. In this embodiment, component I comprises the first protective layer920 with the microwell array 910 comprising an adhesive layer 915 bondedthereto. Component II comprises the second protective layer 930 with theshielding layer 940 bonded thereto. Thus, in this embodiment, the peeladhesion strength of the bond between the first protective layer 920 andthe microwell array 910, the peel adhesion strength of the bond betweenthe microwell array 910 and the adhesive layer 915, and the peeladhesion strength of the bond between the second protective layer 930and the shielding element 940 are all greater than the peel adhesionstrength of the bond between the shielding element 940 and the adhesivelayer 915. In an alternative embodiment (not shown), the relative peeladhesion strengths can be selected such that when the first and secondprotective layers (920 and 930, respectively) are separated, theshielding element 940 remains bonded to the adhesive layer 915 ratherthan the second protective layer 930.

The microwell array 910 is then applied to a component 990 of an opticalsystem, as shown in FIG. 9C. In a preferred embodiment, a peripheralportion (e.g., an edge) of the adhesive layer 915 of the microwell array910 is contacted with the optical component 990 (e.g., a camera, a fiberoptic bindle). The remainder of the adhesive layer 915 is contacted withthe component 990, preferably, by bending the component I into aslightly curved shape and “rolling” the adhesive layer 915 of the curvedflexible microwell array 910 in the direction of the arrow onto thecomponent 990 in a smooth motion to avoid the formation of wrinklesand/or the entrainment of air bubbles between the adhesive layer 915 andthe microwell array 910.

After contacting the microwell array 910 with the optical component 990,the microwell array 910 can be processed to achieve a substantiallyuniform, flat surface on the optical component 990. FIG. 9D shows how,optionally, a roller 995 can be contacted to the exposed surface of thefirst protective layer 920 to provide uniform contact between themicrowell array 910 and the optical component 990. This process canfurther provide more uniform optical properties (e.g., depth of field,depth of focus, adhesive thickness) for imaging each reaction site inthe microwell array 910.

The first protective layer 920 is removed from the microwell array 910to expose the reactive sites for microanalyses, as shown in FIG. 9E.Optionally, a roller may be contacted to the surface of the microwellarray 910, as described above. FIG. 9F shows the microwell array 910optically coupled via adhesive 915 to the optical component 990 formicroanalyses.

Method of Detecting an Analyte

The present disclosure provides methods to detect an analyte. A methodof detecting an analyte comprises providing a sample suspected ofcontaining an analyte, an assay reagent for the optical detection of theanalyte, an optical detection system, and a microwell array articleaccording to the present disclosure. The method further comprisescontacting the sample and the assay reagent in at least one microwellsunder conditions suitable to detect the analyte, if present, in the atleast one microwells. The method further comprises using the opticaldetection system to detect the presence or absence of the analyte in amicrowell.

Suitable conditions include a variety of assay conditions known in theart. Nonlimiting examples of assay conditions include assay conditionsfor the detection of binding partners (e.g., receptor-ligand reactions;antigen-antibody reactions, for example; and nucleic acid hybridizationreactions) as well as assay conditions suitable for an enzyme reaction(e.g., nucleic acid amplification, nucleic acid sequencing). Microvolumeassays performed in microwell arrays are described in, for example, U.S.Pat. No. 6,942,968; and PCT International Publication No. WO 03/016868;each of which is incorporated herein by reference in its entirety.

The assay reagent may comprise a probe (e.g., a protein or apolynucleotide). The probe may be a labeled probe. The probe may belabeled with any of a variety of optically-detectable labels that areknown in the art. The assay reagent may comprise an enzyme. Nonlimitingexamples of enzymes used in optical detection for biological assaysinclude DNA polymerase, thermonuclease, Taq polymerase, and alkalinephosphatase. The assay reagent may comprise an enzyme substrate. Theenzyme substrate may be suitable for a reaction that can be detectedoptically. In some embodiments, the enzyme substrate is a fluorogenicenzyme substrate. In some embodiments, the enzyme substrate is alumigenic enzyme substrate. In some embodiments, the enzyme substratemay react with a first enzyme to produce a product that reacts with asecond enzyme in a fluorometric or lumimetric reaction.

In some embodiments, one or more of the biological assay components ofthe biological assay is attached to a particle (e.g., a microparticle, ananoparticle, a bead). The assay component may be the sample suspectedof containing an analyte (e.g., the analyte is synthesized or capturedand/or concentrated onto a particle). The assay component may be abinding partner (e.g. a polypeptide, antibody, or polynucleotide probethat selectively binds to the analyte). The assay component may be anenzyme. Suitable particle compositions include those used in peptide,nucleic acid and organic moiety synthesis, including, but not limitedto, plastics, ceramics, glass, polystyrene, methylstyrene, acrylicpolymers, paramagnetic materials, thoria sol, carbon graphite, titaniumdioxide, latex or cross-linked dextrans such as Sepharose, cellulose,nylon, cross-linked micelles and Teflon may all be used.

Generally in this embodiment, the particles are non-covalentlyassociated in the microwells, although the microwells may additionallybe chemically functionalized as described herein. Cross-linking agentsmay be used, or a physical barrier may be used, i.e. a film or membraneover the particles, to prevent the particles from migrating out ofand/or between microwells.

The particles need not be spherical. Irregular particles may be used. Inaddition, the particles may be porous, thus increasing the surface areaof the particle available for either bioactive agent attachment oranalyte attachment. The particle sizes range from nanometers, i.e. 100nm, to millimeters, i.e. 1 mm, with beads from about 0.2 micron to about200 microns being preferred, and from about 0.5 to about 5 micron beingparticularly preferred, although in some embodiments smaller beads maybe used.

In some embodiments, each particle comprises a target analyte or abioactive agent, although as will be appreciated by those in the art,there may be some particles which do not contain a target analyte or abioactive agent, depending on the synthetic methods. Alternatively, asdescribed herein, in some embodiments it is desirable that a populationof particles does not contain a bioactive agent or a target analyte.“Candidate bioactive agent” or “bioactive agent” or “chemicalfunctionality” or “binding ligand”, as used herein, describes anymolecule, e.g., protein, oligopeptide, small organic molecule,coordination complex, polysaccharide, polynucleotide, etc. which can beattached to the solid supports of the disclosure. It should beunderstood that the articles of the disclosure have two primary uses. Ina preferred embodiment, as is more fully outlined below, thecompositions are used to detect the presence or absence of a particulartarget analyte; for example, the presence or absence of a particularnucleotide sequence or a particular protein, such as an enzyme, anantibody or an antigen. In an alternate preferred embodiment, themicrowell arrays are used to screen bioactive agents, i.e. drugcandidates, for binding to a particular target analyte.

As will be appreciated by those in the art, the biological agents mayeither be synthesized directly on the particles, or they may be made andthen attached to the particles after synthesis. In a preferredembodiment, linkers are used to attach the biological agents to theparticles, to allow good attachment, sufficient flexibility to allowgood interaction with the target molecule, and to avoid undesirablebinding reactions. In a preferred embodiment, the biological agents aresynthesized directly on the beads. As is known in the art, many classesof chemical compounds are currently synthesized on solid supports,including beads, such as peptides, organic moieties, and nucleic acids.

In a preferred embodiment, the bioactive agents are synthesized first,and then covalently attached to the beads. As will be appreciated bythose in the art, this will be done depending on the composition of thebioactive agents and the beads. The functionalization of solid supportsurfaces such as certain polymers with chemically reactive groups suchas thiols, amines, carboxyls, etc. is generally known in the art.Accordingly, microparticles may be used that have surface chemistriesthat facilitate the attachment of the desired functionality by the user.Some examples of these surface chemistries for microparticles include,but are not limited to, amino groups including aliphatic and aromaticamines, carboxylic acids, aldehydes, amides, chloromethyl groups,hydrazide, hydroxyl groups, sulfonates and sulfates.

In some embodiments, the particles in each well of the microarray maycomprise identical or substantially identical bioactive agents. In analternative embodiment, the microarray may comprise a population ofparticles with related, but nonidentical bioactive agents (e.g.,fragments of a gene or a chromosome). In some embodiments, themicroarray may comprise a population of particles with random bioactiveagents

It should be noted that not all sites of an array may comprise amicroparticle; that is, there may be some sites on the substrate surfacewhich are empty. In addition, there may be some sites that contain morethan one bead.

In general, either direct or indirect detection of the analyte or targetanalyte can be performed. “Direct” detection, as used herein, involvesthe detection of a signal (e.g., a, fluorescent or luminescent signal)as a result of a direct interaction (e.g., binding) of the analyte witha binding partner (e.g., an antibody, a polynucleotide) or a reactioncomponent (e.g., an enzyme or an enzyme substrate). Non-limitingexamples of direct detection include the detection of a labeled (e.g.,fluorescently labeled) antibody that selectively binds to a targetanalyte, the detection of a labeled polynucleotide that selectivelyhybridizes to a target analyte, and the detection of an enzyme product(e.g., a chromatic, fluorescent, or luminescent product) resulting froma reaction of the enzyme with the target analyte. Methods for labelingvarious binding partners and methods for detecting the labeled moleculesare well known in the art.

“Indirect” detection involves, for example the detection of a signal(e.g., a chromatic, fluorescent, or luminescent signal) that isgenerated as a result of the interaction (e.g., binding) of the analytewith an unlabeled “primary” binding partner (e.g., an antibody, apolynucleotide), followed by the interaction of the primary bindingpartner with a labeled “secondary” binding partner. In some embodiments,indirect detection may involve the detection of a reaction product(e.g., an enzyme reaction product) or that is generated, for example, asa result of an enzyme-labeled binding partner selectively binding to theanalyte or as a result of a secondary enzyme reaction that is catalyzedby the interaction of the target analyte with a primary enzyme (seeReaction Schemes I and II).

Reaction Scheme I shows a primary enzyme reaction involving twosubstrates (a double-stranded polynucleotide having a single-strandedregion, and deoxyguanosine triphosphate), an enzyme catalyst (DNApolymerase), and two products (a double-stranded polynucleotide to whichthe guanosine dexoyribonucleotide was added, and pyrophosphate (PPi)).Reaction Scheme II shows a secondary enzyme reaction that can resultfrom the reaction shown in Reaction Scheme I. In the secondary reaction,the enzyme (luciferase) reacts with the substrates (pyrophosphate andluciferin) to generate a chemical product (phosphate) and a detectablesignal (light).

In some embodiments, a secondary detectable label is used. A secondarylabel is one that is indirectly detected; for example, a secondary labelcan bind or react with a primary label for detection, can act on anadditional product to generate a primary label (e.g. enzymes), or mayallow the separation of the compound comprising the secondary label fromunlabeled materials, etc. Secondary labels find particular use insystems requiring separation of labeled and unlabeled probes. Secondarylabels include, but are not limited to, one of a binding partner pair;chemically modifiable moieties; nuclease inhibitors, enzymes such ashorseradish peroxidase, alkaline phosphatases, luciferases, etc.

In some embodiments, the secondary label is a binding partner pair. Forexample, the label may be a hapten or antigen, which will bind itsbinding partner. In some embodiments, the binding partner can beattached to a solid support, for example to allow separation of extendedand non-extended primers. For example, suitable binding partner pairsinclude, but are not limited to: antigens (such as proteins (includingpeptides)) and antibodies (including fragments thereof (FAbs, etc.));proteins and small molecules, including biotin/streptavidin; enzymes andsubstrates or inhibitors; other protein-protein interacting pairs;receptor-ligands; and carbohydrates and their binding partners. Nucleicacid—nucleic acid binding proteins pairs are also useful. In general,the smaller of the two components of a binding pair is attached to anNTP for incorporation into a primer. Nonlimiting examples of bindingpartner pairs include biotin (or imino-biotin) and streptavidin.

Generally, in an assay to detect a binding partner (e.g., anantigen-antibody reaction, a receptor-ligand reaction) a samplecontaining a target analyte (whether for detection of the target analyteor screening for binding partners of the target analyte) is added to thearray, under conditions suitable for binding of the target analyte to acorresponding binding partner, i.e. generally physiological conditions.The presence or absence of the target analyte is then detected. As willbe appreciated by those in the art, this may be done in a variety ofways, generally through the use of a change in an optical signal. Thischange can occur via many different mechanisms. A few examples includethe binding of a dye-tagged analyte to the bead, the production of a dyespecies on or near the beads, the destruction of an existing dyespecies, a change in the optical signature upon analyte interaction withdye on bead, or any other optically-detectable event.

In a preferred embodiment, a change in optical signal occurs as a resultof the binding of a target analyte that is labeled, either directly orindirectly, with a detectable label, preferably an optical label such asa fluorochrome. Thus, for example, when a proteinaceous target analyteis used, it may be either directly labeled with a fluor, or indirectly,for example through the use of a labeled antibody. Similarly, nucleicacids are easily labeled with fluorochromes, for example during PCRamplification as is known in the art. Alternatively, upon binding of thetarget sequences, a hybridization indicator may be used as the label.Hybridization indicators preferentially associate with double strandednucleic acid, usually reversibly. Hybridization indicators includeintercalators and minor and/or major groove binding moieties. In apreferred embodiment, intercalators may be used; since intercalationgenerally only occurs in the presence of double stranded nucleic acid,the label will only be detectable in the presence of targethybridization. Thus, upon binding of the target analyte to acorresponding binding partner, there is a new optical signal generatedat that site, which then may be detected.

Alternatively, in some cases, as discussed above, a reporter such as anenzyme generates a species that is either directly or indirectlyoptically detectable.

Furthermore, in some embodiments, a change in the optical signature maybe the basis of the optical signal. For example, the interaction of somechemical target analytes with some fluorescent dyes on the beads mayalter the optical signature, thus generating a different optical signal(e.g., fluorescence quenching).

As will be appreciated by those in the art, in some embodiments, thepresence or absence of the target analyte may be detected using changesin other optical or non-optical signals, including, but not limited to,surface enhanced Raman spectroscopy, surface plasmon resonance,radioactivity, etc.

The assays may be run under a variety of experimental conditions, aswill be appreciated by those in the art. A variety of other reagents maybe included in the screening assays. These include reagents like salts,neutral proteins (e.g. albumin), detergents, etc., which may be used tofacilitate optimal protein-protein binding and/or reduce non-specific orbackground interactions. Also reagents that otherwise improve theefficiency of the assay, such as protease inhibitors, nucleaseinhibitors, antimicrobial agents, etc., may be used. The mixture ofcomponents may be added in any order that provides for the requisitebinding. Various blocking and washing steps may be utilized as is knownin the art.

Optical Systems

Methods of the present disclosure include the use of an optical systemto detect an analyte in a microwell array article. The optical systemcan include an optical device (e.g., a CCD camera, a line scanner) tocapture image data into transformable and/or quantifiable formats. Insome embodiments, the optical device can be optically-coupled to themicrowell array article. The optical system may further comprise anoptical conduit (e.g., a fiber optic bundle) to carry image componentsfrom the microwell array article to an imaging device. The opticalsystem may further comprise a processor. The processor may compriseimage analysis software to analyze the image data.

Optical devices (e.g., CCD cameras) used in the method of the presentdisclosure will permit simultaneous detection of events in one or moremicrowells in a microwell array. The CCD cameras used in detection canbe coupled with additional optics devices including lenses, fiberoptics, image intensifiers, and the like, for amplifying, focusing,reducing or expanding the images produced in the detection methods ofthe disclosure. One skilled in the art will be readily able to determinethe detector and optics required according to the desired screeningmethods and the dimensions of the microwell arrays.

Optical devices include microscopes, such as confocal microscopes, forexample. Confocal microscopes used in methods of the present disclosurewill permit simultaneous imaging of one or more microwells in amicroarray. The confocal microscope will typically be configured forreceiving an array of the present disclosure which is formatted to thedimensions of a microscope slide.

Optical devices also include raster-scanning laser devices, includingthose that utilize photomultiplier tube detection. Exemplary detectioninstruments useful with the microwell array of the disclosure are wellknown in the art and include, for example, the GenePix 40008 ArrayScanner from Axon Instruments, Inc., GSI Scanarray 3000 from GSILuminomics, and the like.

In one embodiment of the disclosure, the microwell array will beattached via a liquid and/or vapor tight seal to a fluid flow device.Such a fluid flow device will permit sample, solvent, probes, rinsingsolutions, reacting solutions, and the like, to flow over the microwellsand the microparticles thereon, if present. Thus, a fluid flow devicewill be capable of carrying out functions such as those disclosed above.Fluid flow devices known in the art include flow cells such as thoseprovided by manufacturers such as Mindrum Precision, Inc., RanchoCucamonga, Calif.; Upchurch Scientific, Oak Harbor, Wash., and 454 LifeSciences, Branford, Conn.

In a preferred embodiment of the disclosure, the microwell array isattached to a fluid flow device and positioned with respect to adetection instrument such as a confocal microscope or a CCD camera insuch a way as to permit numerous iterations of sample loading,detection, rinsing, and the like, without requiring the arraycomposition to be moved or manipulated in any manner beyond fluid flowthrough the fluid flow device.

The hardware used in the apparatus and detection methods of thedisclosure preferably orients the microwell arrays with respect to thedetector optics or to the CCD camera or other optical detection devicesuch that substantially all discrete sites (i.e., 80% or more,preferably 90% or more, more preferably 95% or more) of an array, and,in a preferred embodiment, all arrays (i.e., 80% or more, preferably 90%or more, more preferably 95% or more), can be accurately andsimultaneously detected. The present disclosure provides a microwellarray that can be positioned directly on an optical device for accurate,simultaneous measurement by, for example, all microwells being orientedat the same distance from the optics or detector.

In some embodiments, the article can be optically-coupled to an imagingsystem. In certain embodiments, the imaging system comprises an imagingdevice (e.g., a CCD camera) with an image sensor density of about 10,000image sensors per mm². In certain embodiments, the imaging systemcomprises a fiber optic bundle with a cross-sectional fiber density ofabout 100,000 optical fibers per mm². The fiber optic bundle cantransmit light emitted from a microwell to an imaging device (e.g., acamera). Advantageously, the articles described herein can bemanufactured such that the area comprising the bottom wall of amicrowell corresponds to the area occupied by about 4 individual imagesensors in said camera or about 4 individual optical fibers in saidfiber optic bundle. It will be appreciated that, by minimizing thethickness of the bottom wall and the optically-transmissive flexiblelayer, any light transmitted through the bottom wall of a givenmicrowell will be directed to a limited and/or predetermined number ofimage sensors or optical fibers proximate the bottom wall of themicrowell, as shown in FIG. 3A.

Optical Isolation

The present disclosure provides for arrays of microwells that areoptically isolated in an X-Y plane and optically transmissive in a Zaxis. The microwells can be optically isolated by a variety of meansdescribed herein. In some embodiments, the means for optically-isolatingmicrowells in an array comprises dispersing or dissolving a colorant inthe microstructured layer. In some embodiments, the colorant isdissolved or uniformly dispersed in the material (e.g., plastic polymer)from which the microstructured layer is formed.

The colorant may be a pigment or a dye that absorbs a selectedwavelength or a selected band of wavelengths (e.g., a particular coloror set of colors or the entire spectrum of visible wavelengths) oflight. Nonlimiting examples of suitable colorants include carbon black,fuchsin, carbazole violet, and Foron Brilliant Blue. Suitable colorantsdescribed herein belong to a variety of dye classes. Nonlimitingexamples of suitable dye classes include anthraquinone dyes (e.g.,1,5-bis[(1-methylethyl)amino)]-9,10-Anthracenedione and1-(hexylamino)-4-hydroxy-9,10-Anthracenedione);bis(trifluoromethanesulfonyl)methylene merocyanine dyes (e.g.,4-[4,4-bis[(trifluoromethyl)sulfonyl]-1,3-butadien-1-yl]-Benzenamine,4,4′-[4,4-bis[(trifluoromethyl)sulfonyl]-1,3-butadienylidene]bis[N,N-dimethyl-Benzenamine,and 4-[4-[4,4-bis[(trifluoromethyl)sulfonyl]-1,3-butadien-1-Morpholine);p-(tricyanovinyl) arylamine dyes (e.g.,2-[4-(dibutylamino)phenyl]-1,1,2-Ethenetricarbonitrile); merocyaninedyes (e.g.,2-[(1-methyl-4(1H)-quinolinylidene)methyl]-5-nitro-Benzonitrile); andindoaniline (indophenol) dyes (e.g.,4-[[4-(dimethylamino)phenyl]imino]-1(4H)-Naphthalenone and4-[[4-(diethylamino)phenyl]imino]-1(4H)-Naphthalenone). Numeroussubclasses of merocyanine dyes, with appropriate absorption spectra, aresuitable as colorants according to the present disclosure.

In some embodiments, the colorant can comprise a1-(alkylamino)-4-hydroxy-derivative of 9,10-anthracenedione. Suitablealkyl groups for the 1-alkylamino-4-hydroxy-derivatives of9,10-anthracenedione include, for example, n-hexyl-; 3-methylbutyl-;-decamethylene-(bis); 3-methoxypropyl-; furfuryl-; n-pentyl-;cyclohexyl-; 2-ethylhexyl-; 2-heptyl-; 3-ethoxypropyl-;3-n-butoxypropyl-; 1,1,3,3-tetramethylbutyl-; 3-methyl-2-butyl-;3-dimethylaminopropyl-; and 1,1-dimethylethyl-groups. Advantageously,nonionic 1-(alkylamino)-derivatives of 9,10-anthracenedione that have amelting point less than about 140° C. are generally quite soluble inresin formulations described herein, thereby permitting the attainmentof higher concentrations of the dyes, if needed, in the polymerizedresins (e.g., acrylate resins, acrylic-based resins derived fromepoxies, polyesters, polyethers, and urethanes; ethylenicallyunsaturated compounds; aminoplast derivatives having at least onependant acrylate group; polyurethanes (polyureas) derived from anisocyanate and a polyol (or polyamine); isocyanate derivatives having atleast one pendant acrylate group; epoxy resins other than acrylatedepoxies; and mixtures and combinations thereof).

The synthesis of amino-derivatives of 9,10-anthracenedione is describedin U.S. Pat. No. 2,128,307; which is incorporated herein by reference inits entirety; and in Examples 73-78 herein.

In some embodiments, the colorant may be a pigment or a dye that permitsthe transmission of a selected wavelength or a selected band ofwavelengths (e.g., ultraviolet wavelengths) of light.

In some embodiments, the colorant may be a pigment or a dye that absorbsa selected wavelength or a selected band of wavelengths (e.g., visiblewavelengths) of light and permits the transmission of a differentselected wavelength or a selected band of wavelengths (e.g., ultravioletwavelengths) of light. Exemplary colorants that absorb visiblewavelengths of light and permit the transmission of ultravioletwavelengths of light are fuchsin and violet pigment 9S949D, availablefrom Penn Color, Doylestown, Pa.

Articles of the present disclosure may include a colorant. The colorantmay include a pigment, a dye, a mixture of pigments, a mixture of dyes,or a combination of any two of the foregoing. In some embodiments, thecolorant comprises a nonionic colorant.

Resin compositions of the present disclosure can include a colorant. Insome embodiments, the resin composition can be polymerized using actinicradiation (e.g., u.v. and/or near-u.v. wavelengths of light). Excessiveabsorption of the actinic radiation can reduce the efficiency of thepolymerization process. Thus, in some embodiments, it may be desirableto select a colorant that does not substantially affect thepolymerization process of the resin composition (e.g., a colorant thatis substantially transmissive for u.v. and/or near-u.v. wavelengths).

In certain preferred embodiments, the colorant does not substantiallyinterfere with the flow of the resin composition into and/or onto themicroreplication tool. The present disclosure provides suitableintensely-colored resin compositions that are unimpeded in flow into themicroreplication mold, as compared to the uncolored resin compositions.

Articles of the present disclosure may be used in a microassay to detectchemical and/or biochemical reactions that emit and/or transmit light.For example, articles of the present disclosure may be used to detectlight emitted by a reaction comprising a luciferase enzyme. Variousluciferase enzymes catalyze reactions that emit light in the range fromabout 480 nm to about 615 nm. In order to reduce or prevent lateraltransmission of the emitted light from one reaction well to an adjacentreaction well, the article should comprise a colorant that substantiallyabsorbs light in the range from about 480 nm to about 615 nm or portionsof that range.

Thus, in some embodiments, it is desirable to select a colorant that issubstantially transmissive for one range of wavelengths of light (e.g.,u.v. and or near-u.v. wavelengths) and substantially absorptive ofanother range of wavelengths of light (e.g., ranges of visiblewavelengths).

Further, it is desirable to select a colorant that has a high enoughsolubility in the resin composition to achieve the light absorptiveproperties without substantially degrading the properties of the polymerand/or substantially interfering with a component (e.g., an enzyme, anenzyme substrate) of the microassay. For example, the resin compositioncomprising the colorant may absorb about four times as much light in the480-615 nm range than it absorbs in the 375-450 nm range. Preferably,the resin composition comprising the colorant may absorb more than fourtimes as much light in the 480-615 nm range than it absorbs in the375-450 nm range.

Thus, there are two factors that can guide the selection of a colorantfor use in articles according to the present disclosure: the AbsorbanceRatio (A*), which indicates the ability of the resin/colorant mixture toabsorb the wavelengths of light used in the detection assay (in thisexample, wavelengths of about 550 nm and about 600 nm); and theAbsorbance fraction (F), which indicates the ability of theresin/colorant mixture to transmit the wavelengths of light used to curethe resin composition (in this example, a wavelength of about 400 nm).Broadly, both A* and F can be calculated for any two substantiallynon-overlapping ranges of wavelengths. Both factors are defined belowwith respect to a wavelength (400 nm) that is useful for curing apolymer composition and two wavelengths (550 nm and 600 nm) that fallwithin a range of wavelengths that are useful to detect a reactioninvolving the enzyme luciferin:

A*=(A ₅₅₀ +A ₆₀₀/2 where

A₅₅₀=light absorbance at 550 nm, A₆₀₀=light absorbance at 600 nm, andA=−log(fraction of light transmitted)

F=(2×A ₄₀₀)/(A ₅₅₀ +A ₆₀₀) where

A₄₀₀=light absorbance at 400 nm, A₅₅₀=light absorbance at 550 nm,A₆₀₀=light absorbance at 600 nm, and A=−log(fraction of lighttransmitted)

A higher A* factor indicates that the resin composition comprising acolorant is absorbing more light in the visible light (550-600 nm inthis example) and, therefore, allows less optical cross-talk betweenadjacent microwells in a microassay. A lower F factor indicates that theresin composition is permitting more near-u.v. light (400 nm in thisexample) relative to visible light (550-600 nm in this example) and,therefore allows good curing of the polymer while also absorbing lightin the longer visible wavelengths.

In some embodiments, the colorant in the monomer mixture provides anAbsorbance Ratio (A*) of about 0.3 or greater; preferably, an A* of 0.5or greater; more preferably an A* of 1.0 or greater; even morepreferably an A* of 1.5 or greater, and even more preferably, an A* of2.0 or greater in a microstructured layer with a thickness of 5 microns.In some embodiments, the colorant in the monomer mixture provides anAbsorbance Ratio (A*) of about 0.3 or greater; preferably, an A* of 0.5or greater; more preferably an A* of 1.0 or greater; even morepreferably an A* of 1.5 or greater, and even more preferably, an A* of2.0 or greater in a microstructured layer with a thickness of 10microns. The preferred mixtures allow rapid photopolymerization of theresin composition, while keeping the colorant molecularly dispersed inthe article after polymerization.

In some embodiments, the colorant in the monomer mixture provides anAbsorbance fraction (F) of 0.25 or less, preferably 0.10 or less.

It will be recognized by a person of ordinary skill in the relevant artthat the principles embodied by A* and F can be applied to otherselected wavelengths for photocuring and the Examples described beloware merely exemplary of the way these factors can be used to selectcombinations of colorants with specific desirable attributes fordetecting a luciferase enzyme reaction.

A drawing can illustrate the effects of the length of the optical pathbetween the bottom of a microwell and the image capture device (e.g., acamera or a fiber optic bundle). FIGS. 3A and 3B illustrate severalaspects of the disclosed microwell array articles that limit the amountof optical cross-talk according to the present disclosure.

FIG. 3A shows a schematic view of a microwell array article 300 a, asshown in FIG. 2A, that is optically coupled to an optical device 390. Inthis embodiment, the microstructured layer 310 comprises a colorant thatis substantially nontransmissive to selected wavelengths of light.

The microwell array article 300 a comprises two microwells, 322 a and322 b, formed in microstructured layer 310, and anoptically-transmissive layer 330. The optical device 390 comprises of anarray of sensors 392 (e.g., optical fibers in a fiber optic array orimage sensors in a digital camera). Sensors “P” and “Q” represent twoindividual sensors 392 that are positioned proximate microwells 322 aand 322 b, respectively. Also shown in FIG. 3A are arrows representingoptical signals (e.g., photons) emerging from a biochemical reactionoccurring in each respective microwell.

It can be seen in FIG. 3A that light passing into the relatively thickersidewalls of the microwells does not penetrate far enough through themicrostructured layer 310 to be received by any of the sensors 392. Thisis due to absorption of light by the colorant over the relatively longpath length between the microwells 322 a and 322 b. In contrast, lightpassing into the relatively thinner path length of the bottom walls ofthe microwells 322 can be transmitted through the microstructured layer310 and the optically-transmissive flexible layer 330 and be received bythe adjacent sensors 392. In FIG. 3A, only the light transmitted frommicrowell 322 a is received by sensor “P” and only the light transmittedfrom microwell 322 b is received by sensor “Q”.

FIG. 3B shows a schematic view of a microwell array article 300 b thatis optically coupled to an optical device 390. In this embodiment, themicrostructured layer 310 comprises a colorant that substantiallyabsorbs selected wavelengths of light and the optically-transmissivelayer 330 is relatively thicker than the corresponding layer in FIG. 3A.

The microwell array article 300 b comprises two microwells, 322 c and322 d, formed in microstructured layer 310, and anoptically-transmissive layer 330. The optical device 390 consists of anarray of sensors 392 (e.g., optical fibers in a fiber optic array orimage sensors in a digital camera). Sensors “R” and “S” represent twoindividual sensors 392 that are positioned proximate microwells 322 cand 322 d, respectively. Also shown in FIG. 3B are arrows representingoptical signals (e.g., photons) emerging from a biochemical reactionoccurring in each respective microwell.

It can be seen in FIG. 3B that light passing into the relatively thickersidewalls of the microwells does not penetrate far enough through themicrostructured layer 310 to be received by any of the sensors 392 dueto absorption of light by the colorant over the relatively long pathlength between the microwells 322 c and 322 d. In contrast, lightpassing into the relatively thinner bottom walls of the microwells 322can be transmitted through the microstructured layer 310 and theoptically-transmissive flexible layer 330 and be received by theadjacent sensors 392. In contrast to the microwell array article 300 ain FIG. 3A, some light transmitted from microwell 322 c is received bothby proximate sensor “R” and by distal sensor “S”. Similarly, lighttransmitted from microwell 322 d is received both by proximate sensor“S” by distal sensor “R”.

Optical cross-talk between adjacent microwells can be minimized byseveral factors including, for example, an optically nontransmissivecolorant dispersed in the microstructured layer, an opticallynontransmissive coating on the sidewalls of the microwells, relativelythin bottom walls in the microwells, relatively thinoptically-transmissive flexible layers, increasing the spatialseparation of the microwells, and any combination of two or more of theforegoing factors. Optical cross-talk can also be affected by thedensity and cross-sectional area of the individual image sensors,relative to the cross-sectional area of the individual microwells.

As illustrated above, one particular advantage of the present disclosureis that, particularly through the use of thin optically-transmissivelayers and fiber optic technology, improved extremely high densitymicrowell arrays can be made.

Assay System:

The present disclosure provides an assay system for detecting ananalyte. The assay system comprises a microwell array article asdescribed herein, an optical device, and a processor.

The microwell array article is optically coupled to the optical device.The optical device can be a component of an optical system, as describedherein. The optical device or system is capable of obtaining an image ofat least one microwell. Preferably, the optical device or system iscapable of obtaining simultaneous images of a plurality of microwells.The optical device is capable of providing an image to the processor. Insome embodiments, the processor may obtain the image from the opticaldevice.

The processor may comprise a means for storing an image (e.g., memorysuch as random access memory (RAM), read-only memory (ROM), compact discread-only memory (CD-ROM), non-volatile random access memory (NVRAM),electrically erasable programmable read-only memory (EEPROM), or flashmemory, for example). The processor may further comprise a means fordisplaying an image (e.g., a monitor). The processor may furthercomprise image processing and analysis routines to identify,characterize, and/or quantitate an analyte in a microwell. In someembodiments of the present disclosure, detecting the presence or absenceof the analyte comprises displaying, analyzing, or printing the image ofa microwell.

EMBODIMENTS

Embodiment 1 is a method of detecting an analyte in a microwell array,comprising:

providing

-   -   a sample suspected of containing an analyte;    -   a reagent for the optical detection of the analyte;    -   an optical detection system; and    -   an article, comprising        -   a microstructured layer with upper and lower major surfaces,            comprising a plurality of optically-isolated microwells            extending below the upper major surface; and        -   an optically-transmissive flexible layer coupled to the            lower major surface of the microstructured layer;        -   wherein each microwell in the microstructured layer            comprises an opening, an optically-transmissive bottom wall,            and at least one side wall extending between the opening and            the bottom wall;        -   wherein a thickness (t) is defined by a thickness of the            bottom wall plus a thickness of the optically-transmissive            flexible layer; and        -   wherein t is about 2 μm to about 55 μm;

contacting the sample and the reagent in at least one microwells underconditions suitable to detect the analyte, if present, in the at leastone microwells; and

using the optical detection system to detect the presence or absence ofthe analyte in a microwell.

Embodiment 2 is the method of embodiment 1, wherein the optical systemis optically coupled to the substrate.

Embodiment 3 is the method of embodiment 1, wherein the optical systemcomprises a fiber optic face plate and wherein using the opticaldetection system comprises passing a signal through the fiber optic faceplate.

Embodiment 4 is the method of embodiment 1, wherein the optical systemcomprises a CCD image sensor, a CMOS image sensor, or a photomultipliertube.

Embodiment 5 is the method of embodiment 1, wherein the optical systemfurther comprises a processor.

Embodiment 6 is the method of embodiment 1, wherein detecting thepresence or absence of an analyte comprises detecting light that isindicative of the presence of the analyte.

Embodiment 7 is the method of embodiment 6, wherein detecting lightcomprises detecting light by absorbance, reflectance, or fluorescence.

Embodiment 8 is the method of embodiment 7, wherein detecting lightcomprises detecting light from a lumigenic reaction.

Embodiment 9 is the method of embodiment 1, wherein detecting thepresence or absence of the analyte comprises obtaining an image of amicrowell.

Embodiment 10 is the method of embodiment 9, wherein detecting thepresence or absence of the analyte comprises displaying, analyzing, orprinting the image of a microwell.

Embodiment 11 is the method of embodiment 1, wherein contacting thesample and the reagent in a plurality of microwells under conditionssuitable to detect the analyte comprises an enzyme and an enzymesubstrate.

Embodiment 12 is the method of embodiment 1, wherein contacting thesample and the reagent in a plurality of microwells under conditionssuitable to detect the analyte comprises forming a hybrid between twopolynucleotides.

The invention will be further illustrated by reference to the followingnon-limiting Examples. All parts and percentages are expressed as partsby weight unless otherwise indicated.

EXAMPLES

All parts, percentages, ratios, etc. in the examples are by weight,unless noted otherwise. Solvents and other reagents used were obtainedfrom Sigma-Aldrich Chemical Company; Milwaukee, Wis. unless specifieddifferently.

Materials

3M 8402: a tape obtained from 3M Company, St. Paul, Minn.

3M 8403: a tape obtained from 3M Company, St. Paul, Minn.

Carbon black paste #9B898: 25% carbon black paste obtained from PennColor, Doylestown, Pa.

Darocur 1173: 2-hydroxy-2-methylprophenone obtained from Ciba SpecialtyChemicals, Basel, Switzerland.

Darocur TPO: diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide obtainedfrom Ciba Specialty Chemicals, Basel, Switzerland.

Desmodur W: a diisocyanate, sometimes referred to as H12MDI or HMDI,obtained from Bayer, Pittsburgh, Pa.

Dytek A: an organic diamine obtained from Invista, Wilmington, Del.

EGC-1720: a fluorocarbon solution obtained from 3M Company, St. Paul,Minn.

Fluorescebrite Plain Microspheres: Fluorescent beads obtained fromPolysciences, Inc. Warrington Pa.

Irgacure 819: phenyl-bis-(2,4,6-trimethyl benzoyl)phosphine oxideobtained from Ciba Specialty Chemicals, Basel, Switzerland.

Kapton H: a polyimide film obtained from DuPont, Wilmington, Del.

Loparex 10256: a fluorosilicone treated PET release liner obtained fromLoparex, Willowbrook, Ill.

Lucirin TPO-L: 2,4,6-trimethylbenzoylphenyl phosphinate obtained fromBASF, Luwigshafen, Germany.

Melinex 453: a 25 micron (1 mil) thick polyester film, which is adhesiontreated on one side, obtained from Dupont, Wilmington, Del.

Photomer 6210: obtained from Cognis, Monheim, Germany

Photomer 6602: obtained from Cognis, Monheim, Germany

Scotchcast Electrical Resin #5: a resin obtained from 3M Company, St.Paul, Minn.

SilFlu 50MD07: A release liner available from SilicoNature USA, LLC,Chicago, Ill.

SR238: 1,6 hexanediol diacrylate obtained from Sartomer, Inc., Exton Pa.

SR339: 2-phenoxy ethyl acrylate obtained from Sartomer, Inc., Exton Pa.

SR545: an MQ resin obtained from Momentive Performance Materials,Albany, N.Y.

Teonex Q71: a six micron thick poly(ethylene naphthalate), or PEN, filmobtained from Dupont-Teijin, Chester, Va.

Vitel 1200B: a copolyester resin obtained from Bostik, Wauwatosa, Wis.

Violet 9S949D: a violet paste containing 20% pigment solids obtainedfrom Penn Color, Doylestown, Pa.

Microreplication Tooling

Tooling was prepared by a laser ablation process according to theprocedure discussed in U.S. Pat. No. 6,285,001, which is incorporatedherein by reference in its entirety. Tool A was constructed by coating aurethane acrylate polymer (Photomer 6602) to an approximately uniformthickness of 165 microns onto an aluminum backing sheet as described inUnites States Patent Application Publication No. 2007/0231541, which isincorporated herein by reference in its entirety, followed by ablatingthe coating to produce a hexagonally packed array of posts. Theresulting posts had a center to center distance of 42 microns. Each postcomprised a circular top having a diameter of 27 microns, a sidewallangle of approximately 10 degrees, and a height of 39 microns. Tool Bwas constructed by ablating a 125 micron thick Kapton H polyimide filmto construct posts having a hexagonally packed array of posts. Theresulting posts had a center to center distance of 34 microns and eachpost comprised a circular top having a diameter of 27 microns, asidewall angle of approximately 10 degrees, and a height of 34 microns.Tool C was constructed from Photomer 6602 in the same way as Tool A tomake a hexagonally packed array of posts with center to center distanceof 34 microns. Each post comprised a circular top having a diameter of27 microns, a sidewall angle of approximately 10 degrees, and a heightof 34 microns.

Tooling Surface Treatments

The polymer Tool A was first plasma treated using an apparatus describedin detail in U.S. Pat. No. 5,888,594, which is incorporated herein byreference in its entirety. The polymer tool was mounted onto thecylindrical drum electrode and the chamber was pumped down to a basepressure of 5×10⁻⁴ Torr. Argon gas was introduced into the chamber at aflow rate of 500 sccm (standard cubic centimeters per minute) and plasmaignited and maintained at a power of 500 watts for 30 seconds. After theargon plasma treatment, tetramethylsilane vapor was introduced into thechamber at a flow rate of 360 sccm and the plasma sustained at a powerof 500 watts for 30 seconds. After the plasma treatment intetramethylsilane vapor, oxygen gas was introduced into the chamber at aflow rate of 500 sccm and plasma sustained at a power of 500 watts for60 seconds. The pressure in the chamber during these plasma treatmentsteps was in the 5-10 mTorr range. The plasma chamber was then vented toatmosphere and the treated tool was dipped in EGC-1720 fluorocarbonsolution. The treated tool was heated in an oven at 120 C for 15minutes. Tool C was treated in the same way as Tool A.

The polymer Tool B was plasma treated using an apparatus described indetail in U.S. Pat. No. 5,888,594, which is incorporated herein byreference in its entirety. The polymer tool was mounted onto thecylindrical drum electrode and the chamber was pumped down to a basepressure of 5×10⁻⁴ Torr. Argon gas was introduced into the chamber at aflow rate of 500 sccm and plasma ignited and maintained at a power of500 watts for 30 seconds. After the argon plasma treatment,tetramethylsilane vapor was introduced into the chamber at a flow rateof 360 sccm and the plasma sustained at a power of 500 watts for 30seconds.

Resin Preparation

Resin formulations were prepared as follows.

Solution A: 1125 grams of Photomer 6210, 375 grams of SR238 and 15 gramsof Darocur 1173 were combined in a glass jar. Solution B: 3.75 g ofIrgacure 819 was added to SR339 followed by roller mixing overnight todissolve the Irgacure 819. Solution C: 3.75 grams of Irgacure 819 wasadded to 187.5 grams of SR 238 followed by roller mixing 18 hours todissolve the Irgacure 819. Solution D: solutions A, B, and C werecombined in a glass jar followed by mixing. To this was added Darocur1173 (3.7 g) and Darocur TPO (32 g) followed by roller mixing for 30minutes.

Solution E: Solution D (708 g) was placed in an amber glass jar. Carbonblack paste #9B898 (97 g) was added to the solution and roller mixed for18 hours to provide a resin formulation with a final carbon blackconcentration of 3%.

Solution F: Solution D (466 g) was placed in an amber glass jar. Carbonblack paste #9B898 (40.5 g) was added to the solution and roller mixedfor 18 hours to provide a resin formulation with a final carbon blackconcentration of 2%.

Solution G: Solution D (466 g) was placed in an amber glass jar. Carbonblack paste #9B898 (19.4 g) was added to the solution and roller mixedfor 18 hours to provide a resin formulation with a final carbon blackconcentration of 1%.

Solution H: Solution D (708 g) was placed in an amber glass jar. Violet9S949D (121 g) was added to the solution and roller mixed for 18 hoursto provide a resin formulation with a final violet pigment concentrationof 3%.

Solution I: Into a 500 mL glass jar was placed 99.00 g of SR238 (1,6hexanediol diacrylate) and 10.00 g of SR339. To the solution was added5.94 g of oil blue A (solvent blue 36) and 5.94 g of solvent violet 37and the composition was mixed to disperse/dissolve the dyes. The mixturewas centrifuges and the supernatant (193.85 g) was recovered. 0.68 g ofIrgacure 819 and 3.30 g of TPO-L was added to the supernatant. The jarwas then capped and placed in a shaker for mixing overnight. Most of thedye appeared to be dissolved in the acrylates. Subsequently, to thesolution was added 90.00 g of the base resin with Photomer 6210. Thesolution was subjected to a further mixing in a shaker for 1 hour. Ahomogeneous blue-colored solution was obtained.

Examples 1-5

Microreplication was performed using a UV curing process as described inPCT Publication No. WO 9511464, and described above. Unless notedotherwise, the UV cure process used in these examples did not includethe optional second radiation source described in FIG. 5.

Tool A, having a patterned area of approximately 7 inches by 36 inches,was secured to a mandrel having an approximate diameter of 37 inchesusing 3M 8402 adhesive tape. The Melinex 453 film was threaded from theunwind idler, along the surface of the Tool A, to the rewind idler asshown in FIG. 5. The surface-treated (adhesion-promoting) side of thefilm was facing the tool. The mandrel was heated to 54 C (130 F). Thefilm was run at a line speed of 10 cm/s (20 feet per minute) at a nippressure of 207 kPa (30 psi) at the contact point of the first niproller (a 95 Shore D nitrile rubber roller) and the mandrel. Resin wasapplied to the film by manually pouring a small continuous bead of resinsolution on the film at the hopper location upstream from the mandrel asdepicted in FIG. 5. The resin spread laterally across the width of thetool at the rubber nip roller, forming a bank of solution approximately9 inches wide. Resin solutions E, F, and G were used in Examples 1, 2,and 3, respectively. Resins were cured using radiation from Fusion Dlamps. The Fusion D lamps were operated at an input power of 236 wattsper cm. The cured microwell array film article was removed from the toolat the second nip roller and wound on the rewind idler as shown in FIG.5. Additional samples were made with the above procedure using Tool Binstead of Tool A. Example 4 was made by using Tool B with resinsolution F and Example 5 was made by using Tool B with resin solution H.

Cure depth for several examples were determined using a combination ofSEM imaging and a thickness gauge and are shown in TABLE 1. It can beseen from these examples that increased photoinitiated cure depth can beaccomplished by providing a tooling material that allows greaterpenetration of light (example 1) or alternatively adding a wavelengthspecific colorant with a lower absorbance cross section in thewavelength range of the photoinitiator (Example 5).

TABLE 1 Cure Depth Example Microstructure Cure Number Resin SolutionTool Depth (microns) 1 F (3% carbon black) A (urethane 39 (full curedepth) acrylate) 4 F (2% carbon black) B (polyimide) about 12 5 H (3%violet pigment) B (polyimide) 34 (full cure depth)

Portions of selected samples were cut and dip coated using ScotchcastElectrical Resin #5. The samples were allowed to cure for at least 24hours before microtoming. The embedded samples were thin sectioned(10-um sections) using a diamond knife. The sections were placed in1.515 RI oil and covered with a cover slip prior to imaging. Sampleswere imaged by optical microscopy. A number of sections (listed as“Count” in TABLE 2) were measured to determine the average thickness ofthe well base (bottom wall), as shown in TABLE 2.

TABLE 2 Thickness of material at the base of the wells in micronsExample 3 Example 2 Example 1 1% carbon (G) 2% carbon (F) 3% carbon (E)Average 0.9 2.2 1.8 Std. Dev. 0.3 0.6 0.4 CV 0.31 0.27 0.22 Minimum 0.41.1 1.2 Maximum 1.4 3.5 3.0 Count 18 24 22

Approximately 1×1 inch samples were obtained from microstructured filmexamples 1-3 and Melinex 453 film. The films were placed on a 1×3 inchmicroscope slide, with a small gap (no film) between the samples.Brightfield transmission images were obtained using a Zeiss AxioPlan 2microscope (Plan-Neofluor 10×/0.03 objective) and a Zeiss AxioPlan 2digital camera (8 bit). Prior to final image acquisition the lightintensity was adjusted to ensure the blank area between the films wasbelow the saturation level of the digital camera. Line scans of eachimage were produced using ImagePro Plus image analysis software (MediaCybernetics) across the “blank” area of the slide (the gap between thefilms), an area of the slide that contained just the Melinex 453 film,and an area of the slide that contained the composite article comprisingthe colorant-containing resin cured on the Melinex 453 substrate. FIG.6A is a drawing of a top view of one of the composite articles ofExample 1, with the path of a linescan shown as dashed line A across thecircular microwells and the area between the microwells. FIG. 6B (line3) shows the pixel intensities of each pixel along the line scan shownin FIG. 6A. Also shown in FIG. 6B are the corresponding line scans forthe “blank” (no film, line 1) and PET film (film only, line 2) images.Pixel intensities from the well bottoms were compared to the pixelintensities of the PET film to estimate the average percent transmissionof light through the bottom walls of the wells. The calculated resultsare reported in TABLE 3. It can be observed from these measurements thatthe thin well base substantially transmits light while the walls aresubstantially non-transmissive.

TABLE 3 Light transmission through well base Example Number %transmission 3 (1% carbon black) 86.9 2 (2% carbon black) 87.9 1 (3%carbon black) 80.2

Lateral light transmission through the sidewalls in the X-Y plane (seeFIG. 1) of Example 1 was estimated by preparing a cured film of uniformthickness similar to the midpoint sidewall thickness in Example 1(approximately 5 microns). A small amount of solution E was applied to apolyester film 1. This was covered with a second film 2 and manualpressure was applied to spread solution E between the films. Thesolution between the films was cured by passing under a UV source (500 Wfusion lamp) at 7.6 cm/s (15 ft/min) with film 1 facing the UV source.Film 2 was removed and the resin adhered to film 1 on the UV-exposedside was washed to remove uncured monomer. Cured resin thickness wasmeasured using a caliper gauge. The mean thickness was determined to be4 microns. A portion of the film containing the cured resin was placedin a spectrophotomer (Tecan Infinite M200). Light transmission at 550nanometers was measured at three locations. For the 4 micron film, amean absorbance value of 1.4 was obtained, corresponding to a lighttransmission of 4%. This example serves to illustrate that themicrostructured wells are substantially transmissive along the Z axisand substantially nontransmissive in the X-Y plane.

Examples 6 and 7

Six micron thick Teonex Q71 film was primed on one side with a 5% solidssolution of Vitel 1200B in an 85%/15% mixture of dioxolane andcyclohexanone via a slot-die coater, followed by drying in an oven at160° F. for 2 minutes. The thickness of the coating was 300 nanometersas measured with a white light interferometer. The film was then coatedon the opposite side with a silicone-polyurea adhesive which consistedof a 28% solids solution of an MQ resin (SR545) and a silicone polyurea(SPU) elastomer at a ratio of 55:45. The SPU elastomer was formedthrough the condensation reaction of a 33 kDa diamino terminatedpolydimethylsiloxane, Dytek A, and Desmodur W in a ratio of 1:1:2, asdescribed in U.S. Pat. No. 6,824,820. The film was then dried in an ovenat 160° F. for 2 minutes and laminated to a PET film by passing thematerial through a nip roll in contact with Loparex 10256 fluorosiliconetreated PET release liner. The thickness of the coating was 4.2 micronsas measured by a white light interferometer.

Example 6 was made by performing microreplication as in Examples 1-5using the coated Teonex Q71 film in place of the Melinex 453 polyesterfilm and by using Tool C and resin solution H. Example 7 was made asExample 6 except that resin solution I was used. In Examples 6 and 7 theVitel 1200B-treated side of the Teonex Q71 film was positioned to facetoward the replication tool.

Light transmission through the well base of the microstructure ofExample 6 was measured as described for Examples 1-3 above. FIG. 7 showsthe results of line scans through a “blank” portion of a slide (line 4),through the adhesive-coated PEN film (line 5), and through the microwellarray article (line 6), respectively.

Example 8

A sample made according to Example 1 was coated with a layer of silicondioxide as follows to produce Example 8. The silica deposition was donein a batch reactive ion plasma etcher (Plasmatherm, Model 3280). Themicroreplicated article was placed on the powered electrode and thechamber pumped down to a base pressure of 5 mTorr. The article wasplasma treated first in an argon plasma at 25 mTorr pressure for 20seconds. Following this, tetramethylsilane vapor was introduced at aflow rate of 150 sccm and plasma maintained at a power of 1000 watts for10 seconds, following which, oxygen gas was added to thetetramethylsilane at a flow rate of 500 sccm with the power maintainedat 1000 watts for another 10 seconds. After this step, thetetramethylsilane vapor flow rate was decreased in a stepwise mannerfrom 150 sccm to 50 sccm, 25 sccm and 10 sccm while the plasma was stillon and each of these steps lasted for 10 seconds. After the last step oftetramethylsilane vapor flow of 25 sccm, the flow was disabled and a 2%mixture of silane gas in argon was introduced instead at a flow rate of1000 sccm with the plasma maintained at 1000 watts and treatmentperformed for another 60 seconds. The plasma chamber was subsequentlyvented to atmosphere and the plasma treated microreplicated article wasremoved from the chamber.

Examples 9 and 10

Microwell array articles were prepared by casting and curing solution Eonto a 25 micron (1 mil) PET film as in Example 1. The PET side wasexposed to a solution of potassium hydroxide (40%) containingethanolamine (20%) to chemically etch the PET film. Etching wasaccomplished placing the microstructured side of a section of film(about 7.6 cm (3 inches) by 10 cm (4 inches)) against a sheet of printedcircuit board material. The perimeter of the film was sealed against theboard using 3M 8403 tape to prevent exposure of the solution to thestructured side. The potassium hydroxide/ethanolamine solution wasplaced in a large glass container and heated to 80 C using a water bath.The boards with adhered films were immersed in the bath for a specifiedtime followed by washing with water. Films etched for 3 minutes had 12microns of remaining PET (Example 9). Films etched for 6 minutes and 10seconds had 5 microns of PET remaining (Example 10).

Examples 11-14

A silicone adhesive was coated onto a liner at various thicknesses. Theadhesive consisted of a 28% solids solution of an MQ resin (SR545) and asilicone polyurea (SPU) elastomer at a ratio of 55:45. The SPU elastomerwas formed through the condensation reaction of a 33 kDa diaminoterminated polydimethylsiloxane, Dytek A, and Desmodur W in a ratio of1:1:2, as in U.S. Pat. No. 6,824,820. The liner used was SilFlu 50MD07which uses a fluorosilicone release chemistry on clear, 50 micron (2mil) PET. The adhesive was coated using a knife coater with a 50 micron(2 mil) wet gap. The adhesive was diluted with toluene to achievevarious thicknesses. The coated liner was dried in an oven at 115° C.for six minutes.

The adhesives were then laminated to samples of microwell array articlesformed on PET film according to Examples 8, 15 and 16 using a rubberroller. The well structures were protected from damage with a PET film,which was then discarded. Example 11 was made by laminating 39 micronthick adhesive to the microwell array of Example 1, which had a PET filmthickness of 25 microns, for a total base thickness of 64 microns.Example 12 was made by laminating 7 micron thick adhesive to themicrostructure of Example 1, which had a PET film thickness of 25microns, for a total base thickness of 32 microns. Example 13 was madeby laminating 3 micron thick adhesive to the microstructure of Example9, which had a PET film thickness of 12 microns, for a total basethickness of 25 microns. Example 14 was made by laminating 2 micronthick adhesive to the microstructure of Example 10, which had a PET filmthickness of 5 microns, for a total base thickness of 7 microns.

To simulate an optical assay coupled to a detection device via a fiberoptic face plate, light spread was measured as function of total basethickness below the microstructure (i.e., the base thickness includedboth the PET film plus the adhesive layer). After etching andapplication of adhesive, sections of films were applied to a fiber opticface plate (6 micron fiber diameters, 47A glass, Schott North America).Approximately 20 μl of aqueous solution containing approximately 1000fluorescent beads (27 micron Fluorescebrite Plain Microspheres) wasplaced on the microstructured side of the laminated film. Beads wereallowed to settle into the base of the microstructured wells by gravity.After the water was allowed to evaporate the laminated film/face plateassembly was placed in a fluorescence microscope (Zeiss AxioPlan 2microscope, Plan-Neofluor 10×/0.03 objective, with fluorescein filterset) with the microstructure side facing down (away from the objective).The microscope was focused on the back side of the face plate. Images ofthe back side of the faceplate were acquired using a fluorescein filterset. The degree of light spread was approximated by counting the numberof 6 micron fibers across the diameter of the fluorescent areasprojected on the face plate. The results are shown in TABLE 4. It can beseen from this data that minimization of the base layer thicknessdecreases the amount of lateral light spread, which in turn minimizesoptical cross talk between neighboring wells.

TABLE 4 Approximate projected diameter of 27 micron beads Base ThicknessNumber of 6 micron Approximate Projected (PET + fibers across diameterof 27 micron Example adhesive) diameter of pro- bead on faceplate Number(microns) jected bead image (microns) 11 64 11 66 12 32 8 48 13 15 6 3614 7 5 30

Examples 15-72

Test for Absorbance fraction (F) of a dye: A double-beam spectrometeroperative from 350 nm to 750 nm, furnished with 10.00 mm cells andspectrophotometric grade ethyl acetate was used in these Examples. Thereference cell contained ethyl acetate. A very small amount of a dye,dissolved in ethyl acetate, and providing a peak absorbance between 0.5and 2.0 was placed in the sample cell. The absorbance was measured at400 nm, 550 nm, and 600 nm. The F fraction was calculated as2×A₄₀₀/(A₅₅₀+A₆₀₀). This ratio is a strong indicator of usefultransmittance in the 375-450 nm region.

Test for coloration of a monomer mixture comprising a colorant: Themonomer mixture (50 wt % 1,6-hexanediol diacrylate containing 0.1% TPOphotosensitizer+50 wt % 6210 photomer) was combined with 1 to 5 wt % ofeach dye in a closed vessel with good mechanical mixing (as by rollingor end-to-end inversion). Mixing was done for 24-72 hours. The mixingvessel was subjected to centrifugation to settle undissolved material.Without separation from the pellet (if present), 1% or less of thesupernatant was withdrawn and diluted 1:1000 in ethyl acetate or monomermixture (as indicated in Table 5). The A* ratio and F ratio werecalculated as described above and are shown in Tables 5 and 6.

Table 5 shows the pertinent data for a wide variety of dyes havingabsorptions in the 500-650 nm range. The commercially-available dyes arelisted according to the generic names listed in the Color Indexpublished by the Society of Dyers and Colourists and the AmericanAssociation of Textile Chemists and Colorists. Anthraquinone9,10-anthracenedione) dyes are designated “Q”. Non-anthraquinone dyesare typically classified as “azo dyes” or the like in the Colour Index.

TABLE 5 Commercially-available dyes absorbing in the 500-650 nm region.The listed dyes are soluble in ethyl acetate (E) and/or in the monomermixture (H). Spectral Lambda max Lambda #2 Range, nm Example GenericName solvent (nm) (nm) 50% max Anthraquinone A* F 15 Solvent Blue 16 E641 594 554-659 Q .61 .12 16 Solvent Blue 30 E 625 590 544-658 Q .63 .2417 Solvent Blue 36 E 639 594 566-656 Q .58 .09 18 Solvent Blue 74 E 643595 560-660 Q .53 .17 19 Solvent Blue 98 E 646 597 569-663 Q .52 .18 20Solvent Blue 102 E 641 594 563-658 Q .50 .12 21 Solvent Violet 11 E 544583 498-600 Q .63 .05 22 Solvent Violet 13 E 584 560 510-631 Q .82 .1723 Solvent Violet 14 E 535 — 472-586 Q .62 .11 24 Solvent Violet 16 E560 614 524-630 Q .71 .09 25 Solvent Violet 37 H 591 551 510-611 Q .49.07 26 Disperse Blue 354 E 602 — 553-638 .79 .05 27 Disperse Violet 5 H541 — 484-584 .50 .42 28 Disperse Violet 10 H 511 — 450-564 .50 .24 29Disperse Violet 11 H 545 583 499-600 Q .48 .04 30 Disperse Violet 17 H520 — 475-571 .08 .15 31 Disperse Violet 26* H 539 577 494-594 Q .33 .0532 Disperse Violet 27 H 550 582 503-620 Q .28 .12 33 Disperse Violet 28H 550 585 504-606 Q .08 .02 34 Disperse Violet 29 H 550 588 501-608 Q.67 .04 35 Disperse Violet 31* E 534 577 495-594 Q .36 .03 36 DisperseViolet 33 E 512 — 449-565 .25 .20 37 Disperse Violet 36 H 530 570491-584 Q .04 .22 38 Disperse Violet 40 H 507 — 445-563 .23 .28 39Disperse Violet 42 H 517 — 459-562 .07 .22 40 Disperse Violet 44 H 540580 490-600 Q .07 .10 41 Disperse Violet 50 H 538 — 482-579 .16 .29 42Disperse Violet 52 H 527 — 466-570 .17 .14 43 Disperse Violet 63 H 545 —493-585 .60 .07 44 Disperse Violet 64 E 527 — 469-580 .06 .10 45 SolventRed 19 E 532 — 473-571 .25 .44 46 Solvent Red 24 E 511 — 452-557 .291.00 47 Solvent Red 27 E 511 — 447-559 .42 1.11 48 Solvent Red 91 E 559— 458-584 .25 .71 49 Solvent Red 127 E 520 553 494-574 Q .45 .16 50Solvent Red 166 E 537 — 465-584 .33 1.07 51 Solvent Red 172 E 528 565480-582 Q .27 .09 52 Disperse Red 5 E 507 — 443-563 .22 .30 53 DisperseRed 8 E 519 — 457-568 .26 .19 54 Disperse Red 9 E 498 — 448-543 .33 .4455 Disperse Red 10 E 480 — 418-537 .23 1.79 56 Disperse Red 11 E 529 566484-582 Q .18 .05 57 Disperse Red 13 E 499 — 438-552 .16 .47 58 DisperseRed 15 E 525 560 476-576 Q .47 .06 59 Disperse Red 21 E 500 — 438-556.25 .46 60 Disperse Red 24 E 503 — 441-559 .23 .32 61 Disperse Red 27 E518 — 459-560 .13 .18 62 Disperse Red 30 E 446 — 436-551 .10 .54

TABLE 6 List of other dyes absorbing in the 500-650 nm region. Thelisted dyes are soluble in ethyl acetate (E) and/or in the monomermixture (H). The dyes classes are anthraquinone dyes (Q),bis(trifluoromethanesulfonyl)methylene merocyanine dyes (S),p-(tricyanovinyl) arylamine dyes (V), merocyanine dyes (M), andindoaniline (indophenol) dyes (I), respectively. Lambda Lambda Range,Spectral max #2 nm Dye Example Chemical Abstract Service Name CAS No.solvent (nm) (nm) 50% max Class A* F 634-[4,4-bis[(trifluoromethyl)sulfonyl]-1,3- 58559-02-7 E 533 — 504-537 S.47 .004 butadien-1-yl]-Benzenamine 644,4′-[4,4-bis[(trifluoromethyl)sulfonyl]-1,3- 149679-67-4 E 531 —499-569 S .26 .004 butadienylidene]bis[N,N-dimethyl-Benzenamine 654-[4-[4,4-bis[(trifluoromethyl)sulfonyl]-1,3- 126942-09-4 E 529 —492-554 S .75 .03 butadien-1-Morpholine 662-[4-(dibutylamino)phenyl]-1,1,2- 63504-26-7 E 517 — 474-547 V .34 .003Ethenetricarbonitrile 67 1,5-bis[(1-methylethyl)amino)]- 9,10-33175-76-7 E 509 — 457-556 Q .32 .26 Anthracenedione 682-[(1-methyl-4(1H)-quinolinylidene)methyl]-5- 63945-43-7 E 553 — 493-597M .36 .10 nitro-Benzonitrile 694-[[4-(dimethylamino)phenyl]imino]-1(4H)- 132-31-0 E 598 — 489-671 I .55.33 Naphthalenone 70 4-[[4-(diethylamino)phenyl]imino]-1(4H)- 2363-99-7E 580 — 503-626 I .36 .16 Naphthalenone 711-(hexylamino)-4-hydroxy-9,10- 63768-04-7 H 557 596 503-617 Q 2.38 .14Anthracenedione 72 Intratherm Brilliant Blue 308 E 582 — 474-660 .78 .30(available from Crompton Corporation, Middlebury, CT)

Example 73 Preparation of 1-n-hexylamino-4-hydroxy-9,10-anthracenedione

The reaction was run on a magnetic-stirring hot plate in a 125 mLErlenmeyer flask fitted with a reflux condenser and with a PTFE-coatedmagnetic stirring bar. In it was placed Quinizarin, 6.06 g. (25 mmol),potassium carbonate 0.11 g. (0.8 mmol), manganous chloride tetrahydratecatalyst 0.44 g (2.2 mmol), and to this was added 75 g.1-methoxy-2-propanol, b.p. 119 C, containing 5.06 g. n-hexylamine (50mmol). The reaction mixture was heated to reflux with magnetic stirringfor 6 hours. Then dilution with water precipitated the dye productwhich, after decantation of the liquid, steeping in hot water, anddrying, weighed 7.39 g. From this, 5.48 g. was placed in a filterthimble in a Jacketed Soxhlet Extractor, and in the 250 mL boiling flaskwas placed n-pentane. Heating caused cycling every 5 minutes for 68hours (over 750 cycles). The boiling flask contained a dark bluishsolution of 1,4-bis(hexylamino)-9,10-anthracenedione and some of thedesired product. The solution was decanted off a dark crystalline solidproduct, which was washed once with pentane and dried (weight 2.88 g.indicating 52% yield) The solid had a capillary melting point 77 to 79C, indicating good purity. The compound was tested for Absorbancefraction (F) and coloration (A*) as described for Examples 15-43 and theresults are reported in Table 7.

Example 74 Preparation of1-(3-methylbutylamino)-4-hydroxy-9,10-anthracenedione

In a 125 mL Erlenmeyer flask modified with a sampling port was placedQuinizarin, 6.06 g (25 mmol); NaBH₄, 38 mg (1.0 mmol);3-methylbutylamine, 2.18 g (25 mmol); piperidine, 2.13 g (25 mmol); and1-methoxy-2-propanol, 60 g, and the reaction was run as in Example 1. Itwas sampled at 0.5, 0.75, and at the 3.0 hr termination. The sampleswere subjected to TLC using toluene as the eluent. Quinizarin (0.64R_(f)) was observed only in the 0.5 hr sample. The desired product (0.57R_(f)) appeared fully formed at 0.75 hr. The reaction mixture wasdrowned in 700 mL hot water containing 2.50 g (25 mmol) conc. HCl toneutralize the piperidine. Secondary amines appear unreactive insubstitution on Quinizarin. The precipitated dye product, aftercoagulation by concentration, was steeped twice in 900 mL portions ofhot water. The dried crude oily product was chilled to enable shatteringthe product and transferring it to a filter thimble, where it wasdistributed between layers of cotton to minimize aggregation. Extractionby petroleum ether in the Jacketed Soxhlet apparatus was run for 200cycles, the boiler was cooled, and the dark purple solution was decantedoff. The crystallized solid product was washed with petroleum ether anddried. (weight 3.84 g, indicating 50% yield), The capillary meltingpoint of the product was 86-89 C. The compound was tested for Absorbancefraction (F) and coloration (A*) as described for Examples 15-43 and theresults are reported in Table 7.

Example 75 Alternative preparation of1-(3-methylbutylamino)-4-hydroxy-9,10-anthracenedione

1-(3-methylbutylamino)-9,10-anthracenedione was also prepared by analternative procedure without the use of NaBH₄ catalyst. In thisalternative procedure, the procedure of Example 74 was followed withthese changes; no NaBH4, and to remove possible weakly catalyticreagents, 1,2-diethoxyethane of similar b.p. to replace1-methoxy-2-propanol, and 1-methylmorpholine (25 mmol) to replacepiperidine. The reaction was sampled at 2, 18, 24, 48, and at the 70 hrtermination, at which time the TLC showed no Quinizarin. This showedthat the NaBH4 accelerated the reaction as much as 100-fold. The desiredreaction product (55% yield) was recovered as in Example 74. Thecapillary melting point was 86-87 C.

Example 76 Preparation of1-(3-methoxypropylamino)-4-hydroxy-9,10-anthracenedione

The procedure of Example 74 was followed, but with replacement of3-methylbutylamine by 3-methoxypropylamine, 2.23 g (25 mmol), and of thepiperidine by 1-methylmorpholine (25 mmol). The reaction was set to runon a timer for 15 hr without sampling. When drowned in 700 mL water acrystalline precipitate formed. After steeping as in Example 74 anddrying, followed by Jacketed Soxhlet extraction with petroleum ether for1100 cycles, the crystalline solid product was recovered (weight 5.23 g,67% yield). The capillary melting point was 120-121 C. The compound wastested for Absorbance fraction (F) and coloration (A*) as described forExamples 15-43 and the results are reported in Table 7.

Example 77 Preparation of1-(n-butoxypropylamino)-4-hydroxy-9,10-anthracenedione

By the procedure of Example 74 but in a similar 50 mL Erlenmeyer flaskwas placed Quinizarin, 2.43 g (10 mmol); NaBH4, 30 mg (0.8 mmol);3-(n-butoxy)propylamine, 1.31 g (10 mmol); morpholine, 0.87 g (10 mmol);1,4-dioxane, 18 g: and (2-methoxyethyl) ether, 6 g. The mixture wasrefluxed with magnetic stirring. Samples for TLC were taken at 1, 2, 4,and 6 hr (termination) as in Example 74 except that dibutyl ether (99%)was used as the eluent. The purple product had an R_(f) of 0.52. Only atrace of Quinizarin (R_(f) of 0.84) was observed in the 6 hr sample.Drowning, steeping, drying, and Jacketed Soxhlet extraction with pentaneas in Example 2 gave the desired the desired product in solution which,upon drying, resulted in an oil product (2.17 g, 61% yield) which latercrystallized to a waxy solid having capillary melting point 46-50 C, andDSC melting point 48 C as shown in Table IV. The compound was tested forAbsorbance fraction (F) and coloration (A*) as described for Examples15-43 and the results are reported in Table 7.

Example 78 Preparation of1-(2-ethylhexylamino)-4-hydroxy-9,10-anthracenedione

The procedure of Example 74 was followed, but with replacement of theamines by 2-ethylhexylamine, 3.23 g (25 mmol) and morpholine, 2.18 g (25mmol), and of the 1-methoxy-2-propanol by 1,2-diethoxyethane, 60 g. TheNaBH4 was solubilized by 15-Crown-5, 0.26 g (1.18 mmol). TLC sampleswere taken at 1, 2, 4, and 8 hr (termination). As in Example 74 thereaction mix was drowned, steeped, and dried (8.21 g) and subjected toJacketed Soxhlet extraction with petroleum ether for 180 cycles. As inExample 76, the desired product remained in the extraction solvent, andwas recovered as an oil (5.73 g, 65% yield), density 30 C/30 C 1.1680,greater than the monomer mixture (see Examples 15-543 for a descriptionof the composition of the monomer mixture) at 1.0608, and enabling (ifin excess) separation by centrifugation for solubility determination andfor spectrophotometry. The constancy of the ratios of the characteristic558 nm peak to the weight % of the dye (14.34, 14.95, and 14.56) provescomplete solubility up to 13 wt %, providing A* of at least 1.8 for a 5micron thickness of the monomer mixture or its corresponding polymer.Solubility of 1-(2-heptylamino)-9,10-anthracenedione to at least 13 wt%, with A* at least 2, was shown similarly. The compound was tested forAbsorbance fraction (F) and coloration (A*) as described for Examples15-43 and the results are reported in Table 7.

Examples 79-88

A number of 1-(alkylamino)-4-hydroxy-9,10-anthracenedione compounds weretested for Absorbance fraction (F) and coloration (A*) as described forExamples 15-43 and the results are reported in Table 7. Table 8 reportsthe physical properties (melting point, heat of fusion, and glasstransition temperature of the 1-(alkylamino)-9,10-anthracenedionecompounds of examples 73-88. Mass spectra were consistent with thestructures listed in Table 8. Melting points were determined by DSC (10°C./minute) with the temperature of maximal heat flow reported. Heat offusion was determined by DSC (10° C./minute). Glass transitiontemperature was determined by DSC (10° C./minute) with the midpoint ofthe transition temperature reported.

Tables 7 and 8 show data from a preferred class of 9,10-anthracenedionedyes for organic media, including polymers includingpolytetrafluoroethylene, for example. All of the dyes absorb strongly inthe 500-650 nm region, as shown in Table 7 by A*, and relatively muchless in the 400 nm region, as shown by F. In each case, except as noted,concentrated dye solutions in a monomer mixture (the composition isdescribed in Examples 15-43) are precisely diluted in ethyl acetate forspectrophotometry. These listed A* values are not upper limits exceptfor the dyes having melting points above 140 C, for which the solutionswere proved by centrifugation to be saturated.

The double peak at 555 and 595 nm is characteristic for1-alkylamino-9,10-anthracenedione, but gives almost no information aboutthe alkyl group. The molar absorbance is the absorbance, in a 1 cm pathlength, of a 1M solution. The molar absorbance reported in Table 7 isreported for the absorbance of the solution at the 555 nm peak. For thepurest dyes of Table 7 it averages approximately 10910+/−180. By usingthis value a dye sample (e.g., a sample of a monomer mixture or apolymer) can be assayed for maximal molar content of a dye of this class

TABLE 7 Spectrophotometric Data for Nonionic1-(Alkylamino)-9,10-anthracenedione dyes highly soluble in an exemplarymonomer mixture that can be used to make microwell array devices. LambdaMolar Abs. Chemical Abstract Spectral max Lambda #2 Range, nm (555 nm)Example Service (CAS) Name CAS No. solvent (nm) (nm) 50% max A* F (×10E4) 73 n-Hexyl- 63768-04-7  E^(a) 555 596 505-613 0.90 0.05 1.135 743-Methylbutyl- E 556 596 507-615 0.85 0.06 1.076^(c) 76 3-Methoxypropyl-93982-26-4  E^(b) 555 593 502-613 0.42 0.08 1.095 ^(d) 773-n-Butoxypropyl- E 555 595 504-615 0.73 0.05 ND 78 2-Ethylhexyl-94023-27-5 E 558 599 505-619 1.77 0.08 ND 79 Decamethylene- (bis) E 555596 493-617 0.02 0.12 ND 80 Furfuryl- E 517 550 450-597 0.13 0.17 ND 81n-Pentyl- 63768-03-6 E 555 596 505-614 1.22 0.07 ND 82 Cyclohexyl-82206-33-5 E 558 597 510-618 0.22 0.07 ND 83 2-Heptyl- H 559 597 508-6151.50 0.06 1.091 84 3-Ethoxypropyl- E 556 596 507-615 0.88 0.04 ND 851,1,3,3-Tetramethylbutyl- E 560 601 503-620 0.62 0.62 ND 863-Methyl-2-butyl- E 557 599 488-616 0.70 0.15 ND 873-Dimethylaminopropyl- 38866-17-0 E 552 591 502-608 0.19 0.04 ND 881,1-Dimethylethyl- E 557 596 364-615 0.34 0.94 ND ^(a)A solution of thisdye in the monomer mixture gave A* = 2.38, F = 0.14; but the sample,capillary MP 63-70 C., was less pure. ^(b)A solution in ethyl acetategave A* = 1.670, F = 0.05. ^(c)Average of 3 measurements; 1.066, 1.078,and 1.093. ^(d) Measured on the ethyl acetate solution of note (b).

In Table 8, the melting points of crystalline solids are taken as thetemperatures of maximal heat flow at heating rates of 10 C per minute inDifferential Scanning calorimetry (DSC), which also provides heats offusion and (on reheating) glass transition temperatures (Tg). Oils andglasses yield only Tg information. Visual capillary melting points ofalmost-black dyes are very imprecise, but are closest for purestsamples.

All high-resolution mass spectrometry (MS) was performed using anAgilent 6540 Ultra High Definition Q-TOF LC-MS/MS instrument (AgilentTechnologies, Inc., Santa Clara, Calif.). The results are reported inTable 8 as calculated (Calc.) values and observed (obs.) formula weight(FW) values.

All thin-layer chromatography (TLC) was performed using Whatman (GEHealthcare Life Sciences Ltd.) MK6F silica gel plates and toluene eluent(unless noted otherwise). Unreacted Quinizarin was detected by itsyellow-orange fluorescence using UV or blue illumination. Desiredproducts were observed as purple spots and1,4-bis(alkylamino)anthraquinone by-products were observed as cyan-bluespots.

TABLE 8 Physical properties of nonionic1-(Alkylamino)-9,10-anthracenedione dyes that are highly soluble in amonomer mixture used for microwell array articles. Melting FW FW^(d)FW^(e) Chemical Abstrate Point H(f) T_(g) (MS) (MS) (Chem.) ExampleService Name (° C.) (J/g) (° C.) (Calc.) (Obs.) (Calc.) 73 n-Hexyl-  7887.8 −21 323.1513 323.1519 323.29 74 3-Methylbutyl-  85 84.2 −20309.1357 309.1358 309.36 76 3-Methoxypropyl- 123 94.0 −20 311.1149311.1151 311.32 77 3-n-Butoxypropyl-  48 44.6 −34 353.1619 353.1623353.40 78 2-Ethylhexyl- Oil ND −27 351.1826 351.1822 351.45 79-Decamethylene-(bis)-  149^(a) 62.5 −15 616.2565 616.2568 616.70 80Furfuryl-  174^(b) 74.0 −14 319.0836 319.0838 319.32 81 n-Pentyl-  8176.0 −19 309.1357 309.1361 309.36 82 Cyclohexyl- 165 90.3 −13 321.1357321.1359 321.36 83 2-Heptyl- Oil ND −17 337.1670 337.1676 337.42 843-Ethoxypropyl-  81 69.5 −28 325.1306 325.1302 325.35 85 1,1,3,3- 138^(c) 10.3 43 351.1826 352.1829 351.45 Tetramethylbutyl- 863-Methyl-2-butyl- 103 80.5 ND 309.1357 309.1360 309.36 87 3-  68 61.2 8324.1466 324.1471 324.38 Dimethylaminopropyl- 88 1,1-Dimethylethyl- 12381.6 ND 295.1200 295.1200 295.35 ^(a)Five trial MPs; 79, 94, 117, 137,and 149 C., observed sequentially. ^(b)Two trial MPs; 83 and 174 C.,observed sequentially. ^(c)Two trial MPs; 83 and 138 C., observedsequentially. ^(d)Mass spectral monoisotopic exact Formula Weights (FW).^(e)Chemical Formula Weights; calculated using natural isotopicabundances of the component elements.

Example 89

The following colorants (listed by their respective generic names fromthe Color Index) were added to the monomer mixture (50 wt %1,6-hexanediol diacrylate) in the weight percent ratio listed below:

Disperse violet 29 4.00% Solvent blue 36 1.5% Solvent violet 11 0.6%Solvent violet 37 0.5% Disperse red 11 0.6% Disperse red 15 0.8%

After thorough mixing, the solution was diluted 1:1000 inspectrophotometric grade ethyl acetate and the absorbance was measuredin a split-beam scanning spectrophotometer (reference solution wasspectrophotometric grade ethyl acetate). The absorbance spectrum of themixture is shown in FIG. 10. The spectrum shows very low absorbance at400-450 nm, an absorbance maximum at about 550 nm, and a secondaryabsorbance maximum (lambda #2) at about 590 nm.

The present invention has now been described with reference to severalspecific embodiments foreseen by the inventor for which enablingdescriptions are available. Insubstantial modifications of theinvention, including modifications not presently foreseen, maynonetheless constitute equivalents thereto. Thus, the scope of thepresent invention should not be limited by the details and structuresdescribed herein, but rather solely by the following claims, andequivalents thereto.

What is claimed is:
 1. A method of detecting an analyte in a microwellarray, comprising: providing a sample suspected of containing ananalyte; a reagent for the optical detection of the analyte; an opticaldetection system; and an article, comprising a microstructured layerwith upper and lower major surfaces, comprising a plurality ofoptically-isolated microwells extending below the upper major surface;and an optically-transmissive flexible layer coupled to the lower majorsurface of the microstructured layer; wherein each microwell in themicrostructured layer comprises an opening, an optically-transmissivebottom wall, and at least one side wall extending between the openingand the bottom wall; wherein a thickness (t) is defined by a thicknessof the bottom wall plus a thickness of the optically-transmissiveflexible layer; and wherein t is about 2 μm to about 55 μm; contactingthe sample and the reagent in at least one microwells under conditionssuitable to detect the analyte, if present, in the at least onemicrowells; and using the optical detection system to detect thepresence or absence of the analyte in a microwell.
 2. The method ofclaim 1, wherein the optical system is optically coupled to thesubstrate.
 3. The method of claim 1, wherein the optical systemcomprises a fiber optic face plate and wherein using the opticaldetection system comprises passing a signal through the fiber optic faceplate.
 4. The method of claim 1, wherein the optical system comprises aCCD image sensor, a CMOS image sensor, or a photomultiplier tube.
 5. Themethod of claim 1, wherein the optical system further comprises aprocessor.
 6. The method of claim 1, wherein detecting the presence orabsence of an analyte comprises detecting light that is indicative ofthe presence of the analyte.
 7. The method of claim 6, wherein detectinglight comprises detecting light by absorbance, reflectance, orfluorescence.
 8. The method of claim 7, wherein detecting lightcomprises detecting light from a lumigenic reaction.
 9. The method ofclaim 1, wherein detecting the presence or absence of the analytecomprises obtaining an image of a microwell.
 10. The method of claim 9,wherein detecting the presence or absence of the analyte comprisesdisplaying, analyzing, or printing the image of a microwell.
 11. Themethod of claim 1, wherein contacting the sample and the reagent in aplurality of microwells under conditions suitable to detect the analytecomprises an enzyme and an enzyme substrate.
 12. The method of claim 1,wherein contacting the sample and the reagent in a plurality ofmicrowells under conditions suitable to detect the analyte comprisesforming a hybrid between two polynucleotides.